NUCLEIC ACID ENCODING AMP-ACTIVATED PROTEIN KINASE

Abstract: cDNAs encoding mammalian AMP protein kinases, corresponding polypeptides and recombinant proteins together with antibodies thereto. Their uses including the study of gene expression and the characterisation of other proteins.

Description: NUCLEIC ACID ENCODING AMP-ACTIVATED PROTEIN KINASE The key enzymes regulating fatty ac d and cnolestercl biosynthesis in tne body are acetyl coA carcoxvlase (ACC) ana KMG CoA reductase (HMG-R) . The release of fatty acids and cholesterol from mtracellular stores of triglyceπdes and cholesterol esters s regulated by hormone sensitive lipase (HSL) . Regulation of these enzymes is multivalent eg feedback inhibition, transcπptional and translational control. More recently, reversible phospnorylation has also been discovered as a mechanism for the acute regulation of at least ACC and HMG-R, and possibly HSL. Work over the past few years has identified the key kmase that regulates the activities of these enzymes. AMP protein kmase, which is itself regulated allosterically -y 5' -AMP and reversible phospnorylation (by a distinct mase-kmase) , can phosphorylate and inactivate ACC and HMG-R (& possibly HSL) . Although agents which inhibit HMG-R, sucn as members of the statin familily of compounds, have found great utility m the ciinic for the treatment of hypercholesterolaemia, there are no acceptable agents for the treatment of combined hyperl pidaemia le elevated fatty acids & elevated cholesterol levels; elevated fatty acids are increasingly being recognised as important in numerous disease states . Carlmg et al (Eur.J.Biochem. , 1989, 186, 129-136) αescπbe tne purification of an AMP protein kmase from rat liver. However, all efforts to clone a mammalian AMP protein kmase appear to nave oeen unsuccessful . According to a first aspect of the present invention MB now provide an cDNA encoding a mammalian AMP protein kmase and convenient fragments thereof. The cDNA is conveniently as set out m Table 1 and a restriction map indicating convenient fragments thereof s sec out m Tac_e 3 I a further aspect of the present invention we provide a recomcmant mammalian AMP protein inase and convenient fragments tnereof . This may for example be obtained by expression of the cDNA as set out m Table 1 or convenient fragments thereof as indicated m Table ; Convenient peptide sequence wnic may be obtained is set out m Taole 2 fet.noάs fcr cDNA expression will be apparent to t.-.e ■ nolεc lar biologist of ordinary sκ ll and include those set out m the Maniatis cloning manual (Molecular Cloning- A Laooratory Manual - 2nd Edition (1989) - J. Sambrook, E.F. Fritsch & Maniatis) , and in Current Protocols in Molecular Biology - 1987 - P. Sharp (Ed.) Particular expression systems include the mouse erythroleukaemia (mel) cell expression system claimed m WO-89/01517 (Grosveld) and, more particularly, as claimed m WO-92-11380 (Hollis et al) . An alternative expression system is the Baculovirus Expression System (Clontech) - A. Prokop et al, Recombmant DNA Technology & Applications, 1991, 97-152. The recombmant mammalian AMP protein kmase of the present invention is used to identify agents which modulate the action of this kmase Such agents are desirable since, for example, they can be used to reduce the biosynthesis of cholesterol and fatty acids. They may also be used to inhibit the release of these from mtracellular stores by HSL. Therefore in a further aspect of the present invention we provide a method for the identification of an agent which increases activation of AMP protein kmase, which method comprises contacting a potential agent with a recombmant mammalian AMP protein kmase and identifying any increase m AMP protein kmase activation over that found m the ansence of the agent. Any convenient assay format may be used. In particular a peptide, for example containing a serme residue exclusively pnospnorylated by AMP protein kmase, is incubated the presence of a preparation of AMP protein kmase and a radiolaoel such as gamma 33-P The reaction is allowed to proceed for a period of about 1 hour and is conveniently terminated by the addition of acid. The phospnorylated peptide is conveniently separated from unincorporated raα oiaoei by omαing to a cnargeo ~emDrar.e followed DV wasn ng Tne degree of phospnorylation of tne peot ce s a measure cf tne act " ty/activation of tne AMP protein Kinase In a further aspect of tne invention we provide a method for the identification of an agent which increases the level of AMP protein Kmase expression, wnich metnod comprises contacting a potential agent with a cell expressing a mammalian AMP protein -: nase and identifying any increase m the level of AMP protein kmase expression over that found m the absence of the agent. This method may involve the use of any convenient in vitro assay. In general, a cell expressing the mammalian AMP protein kmase gene is contacted witn an agent capable of transcriptionally modulating expression of the gene, thereby affecting the level of protein encoded by the gene which is expressed by the cell. Convenient procedures are described in WO-91/01379 (Oncogene) . An increase m transcription may be monitored by blotting techniques using probes derived from the cDNA encoding the AMP protein kmase, or by PCR using primer sequences derived from the cDNA. Protein expression levels can be conveniently monitored by the use of antibodies and enzyme activity measured as described above. The cDNA of the invention encoding a mammalian AMP protein kmase, or any convenient fragment thereof, may be used to provide DNA probes . A convenient probe comprises the full cDNA sequence of a mammalian AMP protein kmase, for example the cDNA sequence set out in Table 1. The molecular biologist of ordinary skill will be aware that the above DNA probes (or RNA probes derived therefrom) may be use in a number of procedures. These include the identification and cloning of homologous mammalian and non-mammalian cDNAs including the associated kmase-k ase. Such homologous cDNAs represent further independent aspects of the invention. They may be cloned into vectors (such as those commercially available) order to produce useful fusion proteins or cloned into expression vectors to construct hign level expressing cell lines . They may also be used in gene cloning studies order to analyse a number of regulatory elements including promoters, ennancers and nitrons. In addition they may oe used to investigate gene expression m "ivo They may also ϋe used n tne preparation of transgen c animals, sucn as mice cr rats As outlined earlier above tne recomomant mammalian AMP protein kmase of the invention, and convenient fragments thereof, may be used to identify agents whicn cause increased enzyme activation. It may be used in molecular modelling and X-ray crystallography studies In aααition t can be useα to map the sιte(s) phosphorylated oy the putative tmase-kmase (which causes activation of AMP protein '.lnase) ana oe-pnosphorylated by a k ase-phospnatase (inactivates AMP protein kmase) This is conveniently acnieved by working out sequentially (1) the number of sites phosphorylated on the protein (11) which sιte(s) are phosphorylated by which protein kmase (m vitro) and ( iu which kmase (s) are responsible for phospnorylation n vivo Baseo on the data derived from the above types of experiments, an AMP protein kmase peptide substrate specific for the ase-kmase and kmase-phosphatase may be developed to allow specific assays for the latter enzymes. The recombmant mammalian AMP protein kmase of the invention, and convenient fragments thereof, may also may be used to raise antibodies Such antibodies have a number of uses which will be evident to the molecular biologist of ordinary skill. These include 1) monitoring protein expression m native cells and clones which express recombmant AMP protein kmase, (11) the development of assays to measure kmase-kmase and kmase phosphatase and (111) the precipitation of AMP protein kmase and other proteins which associate rfith AMP protein kmase leading to identification of these proteins We have found that AMPK shows 47% sequence identity to the protein encoded by the yeast SNF 1 gene, the product of which is essential for release from glucose repression. Whilst we do not wish to be bound by theoretical considerations, AMPK and SNFl may form part of a family of protein kinases that respond to metabolites rather than second messengers We disclose the expression of mammalian AMPK m yeast and structure/function analysis undertaken after m vitro site-directed mutagenesis/gene deletion We also disclose the over-expression of AMPK in yeast and analysis of the effects of increased protein kmase activity on lipid biosynthesis and other cellular processes stuαied in tnis lower eukaryote In addition we disclose tne expression of AMPK as a fusion protein cells of r.epatic origin and tne lαentification of substrates and other proteins /me interact <tfith AMPK tnese cell types, for example using tne procedures disclosed by Yang et al m Science, 1992, 257, 680-682 The invention will now be illustrated but not limited by reference to the following Taoles, Figures and Examples wherein Table 1 shows the full length cDNA sequence of a mammalian AMP protein Kmase. Table 2 shows peptide sequence generated from the above 63kd AMP protein kmase by proteolytic and chemical cleavage. Table 3 snows a linear (six base ) MAPPLOT restriction map of the cDNA sequence of Table 1. Table 4 shows ammo acid sequence information obtained from peptides derived from AMPK. Table 5 shows the full-length cDNA coding sequence of human AMP protein kmase isolated from a skeletal muscle cDNA library. Translational starting codon Met bems at nucleotide residue 2. Table 6 shows a comparison of the full-length cDNA coding sequences of cloned rat and human AMP protein kinases . Figure 1 illustrates the identification of the polypeptide corresponding to AMPK purified from rat liver. (A) The effect of dephosphorylation on the mobility of AMPK polypeptide as determined by SDS-PAGE: AMPK was purified from rat liver up to and including gel-filtration on Superose 12 as previously descπoed (Carlmg et al. , Eur.J.Biocnem, 1989, 186, 129-136) . 10 μg of the preparation was incubated for 30 mm at 30°C either m the absence (Lane 1) or presence (Lane 2) of the catalytic subunit of protein phosphatase 2A. The samples were analysed by SDS-PAGE and protein visualised by staining with Coomassie Lane 3 shows the protein stain of the phospnatase 2A preparation alone The positions of molecular weignt standards are indicated on the right of the figure 'B) In Situ phospnorylation of AMPK. 10 μg of AMPK (as in A) was separated cy electrophoresis on a denaturing 10% polyacrylamide gel and transferred to PVDF memorame Proteins on the blot were suDiecced to an m vitro protein Kmase assay using the etnoo of Celenza and Carlson (Mol.Cell Bic, 1986, __, 5034-5044) The diet was wasneα and autoradiographed at -70°C for 1 hour. The position of molecular weight standards are shown on the right of the autoradiograpn Figure 2 illustrates the generation of a unique oligonucelotiαe probe from tne ammo acid sequence of peptide ELI αeπveα from rat AMPK Primer 1 and primer 3 (as descπoed hereinafter) were used in PCR with rat liver cDNA and an aliquot of the reaction products was used as a template for a second round of amplification using primers 2 and 4 to generate tne nucleotide sequence encoding peptide ELI as described m the Experimental Procedures Figure 3 shows the nucleotide and deduced protein sequences of rat AMPK (A) Nucleot des are numbered on the left and ammo acids on the right. Peptide sequences derived from purified AMPK are underlined. The putative initiation codon and the stop codon are shown in bold type. The nucleotides m the 142 bp deletion (nucleotides 102-216) are shown m italics and the boundaries of the deletion marked by vertical arrows. (B) Schematic diagram of part of the mRNA for AMPK. The coding region is shown boxed and the 5' and 3' untranslated regions are indicated by the solid lines. The position of the 142 bp deletion is indicated by cross-hatched markings. The Bglll and EcoRI restriction endonuclease sites used in contructmg various vectors are also shown. The scale is indicated by the solid bar. Figure 4 illustrates the identification of two transcripts for AMPK mRNA (A) Rat liver cDNA was synthesised from total RNA using random hexamers and used as a template for PCR using AMPK specific primers (see Materials and Methods section) . In a separate reaction, pBS-APK was used as a control . The products of the reaction were analysed by electrophoresis on a 1.2% agarose gel and visualised by staining with ethid um oromide. The position of DNA standards are indicated. (3) The nucleotide and predicted protein sequences of tne AMPK transcript navmg a 142 op deletion is shown ceginning at the presumed initiating metnionme and extending to tne stop codon Ammo ac d residues encoded by the sequence downstream of tne deletion are sno n italics and the stop codon indicated by an asterix Figure 5 shows Northern and Southern Analyses . (A) Northern Analysis Approximately 2 ug of mRNA from the indicated tissues were separated on a 1.2% agarose gel under denaturing conditions, transferred to a charge modified nylon membrane and probed with a 1.9 kb cDNA fragment of AMPK. The blot was washed under stringent conditions (0.1 x SSC, 0.1% SDS, 65°C) and exposed for 5 days at -70°C. The same blot was stripped and re-probed with a l kb fragment of rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . Positions of RNA standards (kb) are indicated. (B) Southern Analysis Approximately 10 μg of rat genomic DNA was digested with the restriction endonucleases shown, separated by electrophoresis on a 1% agarose gel, transferred to a nylon membrane and probed with the entire insert from pBS-APK. The blot was washed under stringent conditions (0.1 x SSC, 0.1% SDS, 65°C) and exposed for 2 weeks at -70°C. Figure 6 shows a comparison of AMPK and Yeast SNFl Protein Sequences. The deduced protein sequences of rat AMPK (top) and SNFl (bottom) were aligned using the GAP programme on the University of Wisconsin package, w th a gap weight of 3.0 and length weight of 0.1. Dots indicate gaps introduced to maximise the alignment. Identities are boxed and shaded. Figure " shows the results of immunoprecipitation of AMPK. (A) AMPK was purified from rat liver up to the DEAE-Sepharose step (Carling et al. , 1989, op cit) and incubated with either preimmune serum (10 ul) or 10 μl of the different antisera raised against four synthetic peptides based on the deduced ammo acid sequence of AMPK (cf . Materials and Methods section) (numbering of the antisera refers to the position of the amino acids fo the peptides m the full length sequence, 1 . After 3 hours protein A-Sepharose was added and the mixture incubated for a further hour at 4°C. The immune complex was tnen precipitated by centr fugat cn and AMPK activity remaining m the supernatant fraction determined by the phospnorylation cf tne 'SAMS' peptide (cf . Materials and Methods section) . Activities shown are measured n the presence of 200 μM AMP and are plotted as a percentage of the activity present in the supernatant fraction following precipitation with preimmune serum. 3) In an experiment similar to that described for (A) , AMPK was incubated with either preimmune serum (40 μi) or increasing amounts of antiserum raised against the peptide spanning ammo acids 361-374 of AMPK. The total volume of antiserum added was kept constant by the addition of preimmune serum. The immune complex was precipitated with protein A-Sepharose and AMPK activity was determined m both the supernatant fraction and m the resuspended precipitate. Activities are plotted as the percentage of the activity in the supernatant using preimmune serum alone. (C) Cell lysates from [ S] methionme labelled Sf9 cells infected with recombmant AMPK baculovirus were immunoprecipitated as descrbed in (A) . The immunoprecipitated proteins were analysed by SDS-PAGE, followed by autoradiography. Lane 1, total cell lysate; lane 2, preimmune serum; lane 3, Ab 278/291; lane 4, Ab 361-374; lane 5, Ab 476/489; lane 6, Ab 496/509. Position of molecular weight standards are indicated on the right. Example 1 CLONING OF A CDNA ENCODING AN AMP-ACTIVATED PROTEIN KINASE Purification of AMP-act vated kmase: Purification of the AMP protein kmase from rat liver was performed according to the method disclosed by Carlmg et , al (Eur.J.Biochem. , 1989, 186, 129-136) as far as the Superose-12 gel filtration step. The resulting partially purified kmase preparation was resolved by SDS-PAGE on 10% polyacrylamide gels (Laemmli, Nature, 1970, 227, 680-685) . Derivation of peptide sequences: Three separate preparations of the AMP protein kmase failed to yield N-terminal sequence, wnich suggested that the intact kmase protein has a blocked N-terminus. In order to obtain internal ammo acid sequence, AMP protein kmase was excised from SDS polyacrylamide gels following electrophoresis and cleaved either enzymatically (with S.aureus V8 protease or endoprotemase lys-C) or chemically (cyanogen bromide) Proteolytic digestion was performed during SDS-PAGE by the method of Cleveland et al (J.Biol.Chem. , 1977, 252, 1102-1106) . Following electrophoresis, the peptides were transferred to PVDF membranes, visualised by staining with coomass e and subjected to automated ammo acid sequence analysis. Chemical cleavage was performed by overnight incubation of tne gel slice with Cyanogen bromide 90% formic acid at room temperature. The supernatant was removed and the pellet dried a Speed- ac aspirator. The residue was washed twice with water, dried and resuspended m SDS-gel loading buffer (Davison et al, J.Gen.Virology, 1992, 73, 2709-2713) . Peptides were resolved by SDS-PAGE and suD]ected to sequence analysis on an Applied Biosystems 475 sequencer The acove proteolytic cleavage gave tne following oept ce sequences • Peptide PKO. XLXPENVL (T/L)DA(Q/P)MNAXDADFG(L) SNM Peptide PK1 : (L) KLRPLYDIPYHFEARΞFLR Peptide PK2 : AT(T/L) (F/E) TXΞLVLQREVEVEVESMDKAGNF X= unknown residue ()= uncertainty of residue At a later stage, the 63kd protein (20ug) was also cleaved with cyanogen bromide and additional peptide sequences were generated (see Table 1) . For example, a fragment of approxiamte size 14K was sequenced to give the sequence BT1. A second fragment of 7K was analysed and shown to g ve more than one amino acid residue at each sequencer cycle. This indicates that more than one polypeptide species was present. However, given the deduced cDNA sequence, the peptide sequence data generated was consistent with peptides BT1 and BT2 being present. A 12K fragment was sequenced and gave the data corresponding to BT3. Finally, an 8K fragment was sequenced and gave the data corresponding to BT4. Peptides BT1-BT4 are all preceeded by a methionme residue n the deduced sequence, which is consistent with cleavage by cyanogen bromide. The sequences of the derived peptide were confirmed by comparison to the cloned cDNA (see Table 1 and below) and were also used to confirm the correct reading frame of the cloned cDNA. Using the original peptide sequence information from proteolytic digests (see above) , we concentrated on peptide PKO which shared residue similarity to all protein kmase sequenced to date iunderlined; see Hanks et al, Science, 1988, 241, 42-52. In order to provide unambiguous DNA sequence encoding peptide PKO, messenger RNA was isolated from rat liver and converted to cDNA using a pre-amplificat on kit (Gibco-BRL, according to the manufacturer's instructions • . A number of degenerate cl gonucleotides were synthesiseα oased en the sequence of PKO. These were as follows ιA.A. sequence = ammo acid sequence) : PKA Oligonucieotide Ξ'-NNN CCN GA(A/G) AA(T/C) GTN (C/T)TN (C/T/A) (C/T)N GA-3' A.A. X P E N V L T/L D sequence PKB Oligonucieotide 5' -CCN GA(A/G) AA(T/C) GTN (C/T)TN (C/T/A) (C/T)N GA(T/C) G-3' A. . P E N V L T/L D A sequence PKD Oligonucieotide 5' -CAT (G/A)TT N(G/C) (A/T) NA(G/A) NCC (G/A)AA (A/G) TC NGC-3' A. A. M N S L G F D A sequence PKC Oligonucieotide 5' - (G/A)TT N(G/C) (A/T) NA(G/A) NCC (G/A)AA (A/G) TC NGC (G/A)TC-3' A. . N S L G F D A D seσuence PKA= potential DNA sequence of N-terminal region of PKO in sense orientation PKB= as PKA but nested at the 3'-end ?KD= potential DNA sequence cf C-terminal region of PKO antisense orientation ?KC= as PKD but nested at tne 3 '-end. N= all combinations of nucleotides ()= nucleotide combinations depending on codon degeneracy Ammo acid key is as indicated previously. Both a 1:10 (Reaction A) and a 1:100 (Reaction B) dilution of tne cDNA svnthesied earlier were made and lul of this cDNA was used as template for PCR. The other components in the reaction were 10X Taq Polymerase buffer (Promega,UK) , deoxynucleotide tπphosphates (dNTPs) at a final concentration of lOOuM, Primer PKA and PKD at final concentrations of SOpmoles. The reaction volume was adjusted to 50ul with water and overlaid with mineral oil (Sigma) . The template was o denatured by heating to 95 C for 5 minutes (using the Thermal cycler - o MJ Research) and the sample held at 70 C prior to the addition of 1 unit of Taq Polymerase (Promega) . The reaction constituents were then o o subjected to the following cycling regime : 94 C 2 mms, 55 C 2 mms, o 72 C 2 mms for 40 cycles. The products were analysed on an ethid um bromide stained agarose gel and a visual inspection carried out in order to detect the predicted product size of approximately 70 base-pairs (based on the prediction from the PKO peptide size) . This product size was not visiole. Accordingly, lul from Reaction A (above) and Iμl from Reaction B, together with lul of a 1:100 dilution of these reactions was transferred to a new set of reactions using the conditions as outlined above out carrying out the PCR using the nested set of primers PKB and PKC. The PCR products were once again analysed using ethidium bromide stained agarose gels. A product of the expected size rfas visiole m all samples out strongest using cDNA from the original l 10C dilution Reaction B 1 Template from this dilution was then subsequently used to generate quantitive amounts of PCR product wnich was eiuted from the gel using standard procedures (Sambrook, J. , Fπtscn, E.F. and Maniatis, T. (1989) Molecular Cloning; A laboratory manual (2nd edition) . The PCR product was "polished" (blunt-ended) using T4 polymerase using puclisned procedures (SamorooK et al, op cit) and cloned into Smal-diges ed pBluescript 'Stratagene) using T4 ligase according to the manufacturers instructions (Pharmacia) . The ligation products were plated out onto the appropriate agar media w th antibiotics & chromogenic substrate (Stratagene) . Putative recomomants (white) were identified and plasmid DNAs isolated from seventy two such clones according to a procedure described by Del Sal et al (B otechniques, 1989, 7, 514-519) . The plasmid DNAs were digested with EcoRI and Xbal (which flank the Smal cloning site) and digested DNAs analysed using agarose gels. In total, sixteen clones revealed the expected product size of approximately 150 base pairs (cDNA sequence together with vector sequence internal to the enzymes used in the digestion) . Plasmid DNA from the sixteen clones was denatured according to the method of Hsaio (Nucl.Ac d.Res . , 1991, 19, 2787) and sequenced with a pBluescript vector primer using a T7 DNA sequencing kit (Pharmacia, instructions followed) . Of the sixteen clones, eignt had the same sequence internal to the nested primers used. This sequence was : GCC CAG ATG AAT GCT AAG A ? M N A K The above sequence clarified two points pertaining to the original PKO peptide. Firstly, the amoigu ty surrounding (Q/P) was resolved to snow that tne residue is Proline (P) . Secondly, the X residue was snown to De Lysine (K) . 3ased on the above nucleotide sequence, the following oligonucieotide was synthesised: GAC GCC CAG ATG AAT GCT AAG, end-labelled with T4 kmase (Sambrook et al, op cit) and used as a prone to screen a rat liver cDNA library (Stratagene) . The conditions of hybridisation were 30ng radiciapelled prooe > radiclapeiled using an o igo-labell g K t from Pharmacia; -10 ' cp / ug, 10 " cpm/mi) m 5 X SSPΞ, lOOug - sonicated and denatured o salmon sperm DNA, 2 X denhardts, 0.1% SDS at 50 C for 4 hours. Filters were wasned with 5 X SSC, 0.1% SDS at room temperature for 2 nours followed by 5 X SSC/0.1% SDS at S O C for 30 minutes ano o autoraαiograpny for 18h at -70 C with intensifying screens < for references refer to Sa orook et al, 1989) 6 A total of 1.5 X 10 plaques were screened and eleven hybridising clones were isolated. Plasmids were recovered from the phage by the procedure of in vivo excison as outlined n the manufacturers instructions. Analysis of the inserts by restriction endonuclease mapping revealed that the clones could be divided nto two groups: clones PK-1( ; 8 clones) contained an insert of 2.6kb, 1-8 whilst clones PK-2 ( ; 3 clones) had an insert of ~2.7kb. The inserts from both sets of clones were sequenced on both strands using vector derived primers and subsequently AMP protein kmase cDNA derived primers. Sequence analysis of PK-1 clones revealed an open reading frame coding for 528 ammo acids which included all the peptides listed in Table 1. However, sequence PK-1 did not contain an m-frame initiating methionme residue, nor did it include ammo acid sequences resembling subdomam I found in all other protein kinases (Hanks et al, op cit) . It was therefore concluded that this clone was incomplete at its 5' -end. Clone PK-2 was sequenced and found to be almost identical to the sequence of clone PK-1 but with additional sequence at the 5' -end. The additional 5'-end sequence coded for an m-frame methionme residue in a very good context for translation initiation (Kozak et al, J.Biol.Chem , 1991, 266, 19867-19870) . This was followed by protein sequence highly homologous to protein kmase subdomam I (Hanks et al, op cit) However, further analysis of tnis clone revealed a 142 base pair deletion immediately downstream of the sequence coding for subdomam I as compared to clone PK-1. The deletion leads to the generation of a premature stop codon m the AMP protein kmase protein sequence, resulting in a truncated peptide of on v 34 amino acids. The possible significance of this deletion s unclear In order to constru entire coding sequence cf AMP protein kmase, rat liver cDNA was amplified using primers based on clones PK-1 and PK-2 (forward primer 5' -GCCGAACATGGCTGAGAAG-3' ; reverse primer: TCTTAGCATTCATCTGGGC-3' ) . A product of the expected size was visible on agarose gels and was eluteα from tne gel and cloned nto pBluescript oy the procedure of T.A cloning (Marchuk et al, Nucl.Acids.Res . , 1991, 19, 1154) . Following sequencing cf the cDNA to verify its orientation ano integrity, the recombmant pBluescript was digested with NotI and Bglll to release a fragment of 160 base pairs. This fragment was purified and ligated onto clone PK-1 tnat had also oeen digested with Bglll and NotI. The resulting clone (pBS-APK) was identified cy restriction mapping of plasmid mmipreps and sequenced once again to show clone integrity. This plasmid contains the full length sequence of AMP protein kmase. Introduction to Examples 2-7 AMP-activated protein kmase (AMPK) plays a central role in the regulation of mammalian lipid metabolism (Carlmg et al. , 1987, 1989a, 1991: Hardie et al. , 1989) . AMPK phosphorylates and inactivates acetyl-CoA carboxylase, the enzyme catalysing the first committed step m fatty acid synthesis. AMPK also phosphorylates and inactivates 3-hydroxy-3-methylglutaryl (HMG) -CoA reductase, a key regulatory enzyme in the synthesis of chloresterol and other lsoprenoid compounds. Furtner studies have shown that AMPK phosphorylates hormone-sensitive lipase in vitro (Garton et al. ) , 1989. AMPK, therefore, has the potential to regulate both the syntnesis and breakdown of triglyceπdes and cholesterol esters The sites phosphorylated within acetyl-CoA carboylase oy AMPK nave been identified directly by ammo acid sequencing (Munday et al . 1988; Davies et al . , 1990) as serme-79. serme-1200 and serme-1215 compared to the predicted sequence deduced from rat cDNA clones (Lopez Casillas et al. , 1988. Phospnorylation of these sites has been observed in both isolated adipocytes and in vivo (Havstead et al . , 1S90: Davies et a . , 1992) . Recently, it has oeen shown tnat phospnorviation of serme-"9 can account for most, f not all, cf tne mact vation cf actyi-CcA carooxylase coserved m vitro iDavies et al . , 1990) Furtnermcre, tne degree of pnospnorylat on of serme-79 was snown to increase response to glucagon treatment of isolated nepatocytes (Sim and Hardie, 1988) and glucagon or adrenaline treatment of adipocytes (Haysteaα et al. , 1990) , in parallel with a decrease m the activity cf acetyl-CoA carboxyiase. These results, taKen together with tne fact that no other protein kinases tested to date nave oeen shown to phosphorylate serme-79, argue strongly m favour of a role for AMPK in the control of fatty acid synthesis . In contrast to acetyl-CoA carboxyiase, the role of onosphorylation of HMG-CoA reductase has been much more controversial. HMG-CoA reductase is subject to a number of forms of regulation, and in fact been cited as one of the most regulated enzymes m nature (Brown and Goldstein, 1990) . Although phospnorylation and mactivation of HMG-CoA reductase in vitro was demonstrated over 15 ago (Nordstrom et al. , 1977; Ingebritsen et al. , 1978, 1979; Beg et al. , 1980) the importance of this form of regulation has, to a large extent, been ignored. This was due to the fact that it appeared that the enzyme always existed in a hightly phosphorylated state, despite oemg able to measture changes in the rates of hepatic cholesterol synthesis (Nordstrom et al. , 1977) ; Brown et al. , 1979) . However, recent developments in the isolation of HMG-CoA reductase, notably the use of cold-clamping (Easom and Zamm t, 1984a) , have shown that both pnosphorylation and activity of HMG-CoA reductase change acutely in response to diet and hormonal status ( Easom and Zammit, 1984b, 1987) . The residue phosphorylated within HMG-CoA reductase by AMPK has been identified by ammo acid seqenecemg of the purified, soluble 53 kDa proteolytic form of the enzyme (Clark and Hardie, 1990a) and corresponds to serme-871 in the hamster sequence (Ch n et al., 1984) . Altnougn th s residue can be pnospnorylated by both protein kmase C and Ca " - calmodulm- ependent protein kmase in vitro (Clarke and Hardie. 1990b) there is no evidence to suggest that these kinases play a physiological role in the phospnorylation and mactivation of HMG-CoA reductase (Zammit and Caldwell, 1991) . However, there is good evidence that, under certain conditions, HMG-CoA reductase m isolated hepatocytes is pnospnorylated ~γ AMPK (Gillespie and Hardie, 1992) In addition to acetyl-CoA carcoxvlase and HMG-CoA reductase, AMPK pnospnorylates normone-sensititive lipase (Garton et al, 1989) a regulatory enzyme involved in the hydrolysis of triglycerides and cholesterol-esters (Cooke et al. , 1982) . Hormone-sensitive lipase purified from bovine adipose tissue has oeen used to identify tne residue pnospnorylated by AMPK (Garten et al , 1989) This was found to correspond to serme-565 as predicted by tne sequence deduced from rat cDNA clones (Holm et al. , 1988) Phosphorylation of this site has been observed in vivo (Garton et al. , 1988), and may prevent the activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kmase (Garton et al. , 1989, Garton and Yeaman, 1990) . As well as being involved m the regulation of iipid metabolised enzymes, tnere is some evidence that AMPK may play a wide role in cellular processes . AMPK phosphorylates glycogen synthase and phosphorylase kmase m vitro (Carlmg and Hardie, 1989b) at sites known to be phosphorylated in vivo (Poulter et al, 1991; Heilmeyer, 1991) . However, in the case of glycogen synthase, the site phosphorylated to AMPK can also be phosphorylated by a number of other protein kinases, including cyclic-AMP-dependent protein kmase, and the physiological significance of phosphorylation of this enzyme by AMPK has not oeen fully addressed. AMPK has been purified over 3000-fold from rat liver and its biochemical properties have been examined (Carlmg et al, 1989a) . As its name indicates AMPK is activated by AMP (approximately 5-fold by concentrations of AMP m the micromolar range) , and this activation appears to be extremely specific for AMP since no other nucleotides or nucleotide analogues that were tested had any significant effect on the activity of the enzyme (Carlmg et al. , 1989a) A second form of regulation of AMPK is by reversible onosphorylation. AMPK can ce inactivated by vitro by treatment with purified protein pnospnatases (Carlmg et al , 1987) Demonstration cf pnosphorylation ano activatinon of AMPK has proved difficult, but partially purified preparations of the kmase can be reactivated upon incubation with MgAT? (Carlmg et al. , 1987) This MgATP-dependent reactivation was net α ^ e to an autopnospnoryiation reaction, since more nighly p_rιf eo preparations cf the inase could not oe reactivated in the presence cf MgATP Altncugn cyclic-AMP has been shown to be essential tne sιgna_ transαuct on patnway whereoy glucagon and adrenaline exert their effects on fatty acid synthesis oy the phosphorylation of acetyl-CoA carboxyiase (Haystead et al. , 1990) cyclic-AMP-dependent protein K nase nas no effect en the activity cf AMPK n vitro Davies et al , 1989) Little progress has oeen maαe on the purification and cnaractεπsat on cf tne protein kinase responsible for the phosphorylation and activation of AMPK, althougn it clearly represents an important control point in the regulation of lipid metabolism. We now describe the purification and ammo acid sequencing of AMPK together with the isolation of AMPK cDNA clones . Antioodies directed against synthetic peptides derived from the predicted sequence of the kmase specifically immunoprecipitate the activity cf AMPK from rat liver. The sequence of AMPK shows 47% identity to the protein encoded by the yeast SNF 1 gene (sucrose nonfermentmg) . The protein product of the SNF 1 gene is a protein kinase which has been shown to be essential for the release from glucose repression (Celenza and Carlson, 1986) . We propose that AMPK and SNF 1 may form part of a larger family of protein kinases that repond to the levels of mtracellular metabolites rather than the more classical second messengers, and whose functions include, but may not be limited to, repondmg to metabolic stress. Example 2 PURIFICATION AND AMINO ACID SEQUENCING OF AMPK The polypeptide corresponding to AMPK has been identified previously by SDS-PAGE following covalent labelling of the enzyme with the AT?-analogue, fluorosulphonylbenzoyladenos e (FSBA) (Carlmg et al . , 1989a) A single, labelled polypeptide was observed with an apparent molecular mass of 63 kDa. We have confirmed that this polypeptide corresponds to AMPK and have isolated cDNA clones encoding the kinase. AMPK was purified approximately 3000-fold from rat liver ana tne polypeptide corresponding to the Kmase purified by SDS-PAGE. The identity cf tnis pci'_"peptide was confirmed cy the following criteria 1) Reaction of the kinase preparation with -C-FSBA resulted in specific labelling of a single 63 kDa polypeptide (Carlmg et al. , 1989a, and data not shown) , 2) In some preparations of the kmase the 63 kDa polypeptide migrated as a distinct doublet following SDS-PAGE (Figure 1A: lane 1) . It s likely that this is due to differences m the phosphorylation state cf the enzyme, since it is known that the AMPK is regulated by phosphorylation. Treatment of AMPK with the catalytic subunit of protein phosphatase 2A, which caused almost complete inactivation of AMPK, resulted m a shift in the migration of the upper band of the doublet to that of the lower band (Figure 1A; lane 2) . No changes m the migration of any other bands in the preparation were observed; 3) The same 63 kDa polypeptide was labelled following an in situ 32 phosphorylation assay utilising [T- p] (Celenza and Carlson, 1986) shown in Figure IB; 4) Amino acid sequence derived from the 63 kDa polypeptide showed extensive homology to protein kinase subdomains (Table 4) . On three separate occasions, from two different preparations of AMPK, no sequence was obtained from the intact polypeptide, suggesting that the kinase had a blocked N-terminus. However, several peptides derived by cleavage of AMPK did yield amino acid sequence and these are shown in Table 4. Analysis of the Swiss-Prot data base revealed that the sequence of ELI (and SVl, which is derived from ELI) was similar to sequences found within other protein kinases spanning subdomam VIb-VII (Hanks and Qumn, 1991) and that CB1 was homologous to protein kinase subdomam V. Example 3 ISOLATION OF AMPK CDNA CLONES We were able to obtain a unique DNA sequence encoding a portion of AMPK by PCR using rat liver cDNA and degenerate oligonucieotide primers corresponding to potential sequences encoding peptide ELI: Figure Z '< . 3ased en the sequence cf the amplified product, a umαue oligonucieotide was synthesised and used to screen a rat liver cDNA library. Eleven positive plaques were isolated wnich could be divided into two groups: clones PK-1 and PK-2. Sequence analysis of clone PK-1 revealed a potential open reading frame coding for 528 am o acids wnicn included all of the peptide sequences listed m Table 4 However, tne deduced translated sequence did not contain an in frame initiating methionme residue, nor did it include am o acid sequences resemcl g subdomam I found in all other protein kinases sequenced to date (Hanks and Quinn, 1991) . It was therefore concluded that this clone was incomplete at its 5' end. Clone PK-2 rfas sequenced and was found to be almost identical to the sequence of clone PK-1, and to include additional sequence at the 5' end. The deduced translation of the additional 5' sequence resulted in an m-frame methionme residue which was in a very good context for translation initiation (Kozak, 1987) and was followed by protein sequence highly homologous to protein kinase subdomam I (Hanks and Quinn. 1991) . However, further analysis of PK-2 revealed that it had a 142 base pair deletion, as compared to clone PK-1, immediately downstream of the sequence coding for subdomam I . An overlapping clone (pBS-APK) containing the cDNA encoding the entire deduced protein sequence of AMPK was made from PK-1 and PK-2. The nucleotide sequence, together with the deduced protein translation, of the composite clone is shown in Figure 3A. A schematic representation of the clone, highlighting the deleted sequence is shown in Figure 3B. The first ATG codon of the open reading frame occurs in a good context for translation initiation (Kozak, 1987) , although no stop codons were found upstream. We believe that this is the initiating methionme since the protein encoded by the cDNA has a mass of 62,250 daltons which is almost identical to that observed for the enzyme isolated from rat liver. The open reading frame is followed by 1 kb of untranslated sequence, cut there is no ocvious polvadenvlation signal or poly A tail, wnich, taken m conjunction with the results of the Northern analysis, indicate that the clone is incomplete at the 3' end. Example 4 ALTERNATIVE SPLICING OF AMPK mRNA In order to examine the nature of the 142 bp deletion from PK-2 m more detail, total RNA isolated from rat liver was reverse transcrioed and the cDNA amplified by PCR (RT-PCR) using a sense obligonucleotide near the 5' end of the cDNA (nucleotides 40-56, 5' GAAGATCGGACACTACG 3') and an antisense oligonucieotide downstream of the deleted sequence (nucleotides 286-303, 5' CCTCCAGACACATATTCC 3') . Two products, which differed by 142 bp, were identified (Figure 4A) . These two products were cloned into Bluescπpt and sequenced revealing the shorter product to have the same deletion that was found in the original PK-2 clones. These results indicate the presence of two mRNA transcripts for AMPK, which have been identified in all tissues examined. We have isolated genomic clones for AMPK which span this region and have found that the deleted sequence corresponds to a single exon. The 142 bp deletion leads to the generation of a premature stop codon m the deduced AMPK protein sequence which would result m a truncated peptide of only 34 am o acids (Figure 4B) . Initiation at the next downstream methionme AMPK would produce a protein of approximately 52 kDa. Example 5 NORTHERN AND SOUTHERN ANALYSES Northern blot analysis of mRNA isolated from a numoer of rat tissues and probed w th part of the AMPK cDNA is shown in Fig. 5A. A single transcript of approximately 9.5 kb is observed in all of the tissues except spleen and testis. The relatively large size of the transcript indicates that tne AMPK mRNA contains a long 3' and/or 5' untranslated region, and makes it unlikely that a deletion cf 142 bp would be detected by this method. The highest levels of transcript were found skeletal and cardiac muscle, with only a modest level detectable the liver Although there may be differences m the amount of mRNA loaded vas judged by the level of GAPDH message) this could not fully account for tne differences in tne level of AMPK mRNA we ccserveo We have σuantitated the level of expression cf AMPK message m a numoer of tissues by competitive PCR (Gillilano et al. , 1990) and these results confirm the Northern data. Previously, tne tissue distribution of AMPK indicated that the highest levels of activity were present in the liver, with almost undetectable levels of activity m skeletal muscle (Davies et al . , 1989) . Southern blot analysis cf rat genomic DNA probed with the entire insert of pBS-APK indicates that AMPK is a single copy gene (Figure 5B) . Discussion (Examples 4-5) We have found that the AMPK gene is expressed in a wide variety of rat tissues. By Northern analysis a single band of approximately 9.5 kb was identified in all of the tissues tested, except spleen and testes. RT-PCR revealed two AMPK transcripts which differed by 142 nt. It is unlikely that a difference of 142 nt m such a large transcript would be resolved by Northern blotting. The two mRNA species are the result m differential splicing of a single exon. The full-length mRNA, in which the 142 bp exon is expressed, encodes AMPK as purified from rat liver. Deletion of the 142 bp exon leads to the generation of a premature stop codon which would result m a truncated peptide of only 34 ammo acids. At present, there is no evidence for the existence of such a translation product. This exon is spliced out in a surprisingly high number of AMPK gene products. In most tissues, as much as 40-60% of all AMPK mRNA lacks this exon. Potentially, splicing could represent a powerful mechanism m the regulation of AMPK expression. The observation that splicing of AMPK mRNA occurs to a much lesser extent in skeletal and cardiac muscle than in other tissues would argue in favour of such a regulatory function, and at least suggest that the degree of splicing is tissue-dependen . However, the extent of splicing did not vary markedly liver during a time-course of starvation and refeedmg (data not shown) , although AMPK activity has been reported to change 2-3 fold under these conditions. Therefore, the physiological role of this splicing remains unclear. The aounoance of AMPK mRNA n skeletal muscle was determined to oe Ξ-fo d higner tnan n liver. Moreover, only 15% of tne message m skeletal muscle lacked the 142 bp exon compared to 40% m liver. Therefore, the transcript encoding functional AMPK is at least 7-fold nigner m muscle than in any of tne otner tissues In accordance with this, we have detected relatively hign levels cf AMPK protein m muscle by Western blotting with specific anti-AMPK antibodies. These observations contrast sharply to enzyme activity measurements, which show very little activity in skeletal muscle. Our results imply that muscle AMPK has a low specific enzyme activity compared to the kinase isolated from liver. AMPK is pnospnorylated and activated by a distinct protein kmase m a reaction which is markedly stimulated by AMP. Previously, it has been shown that the high act vty of AMPK isolated from rat liver is a consequence of the increased concentration of AMP, presumably caused by hypoxia during dissection of the tissue. In this the same conditions were employed for the isolation of AMPK from skeletal muscle as from liver, so that similar post-mortem effects would be expected in both preparations. Nevertheless, the muscle enzyme appears to be in a different phospnorylated state than the liver enzyme, suggesting that its low specific activity may be due to lack of phosphorylation at the activatory sιte(s) . Whether this reflects the state of the kinase m vivo, or in an artefact of the isolation from muscle, remains to be established. It should be noted that we used resting muscle for these studies,- possible activation of AMPK by AMP only occurs after a period of sustained muscle contraction. AMPK is thought to play an important physiological role in the regulation of acetyl-CoA carboxyiase and HMG-CoA reductase, the rate-iimitmg enzymes in fatty acid and cholesterol synthesis, respectively. In accordance with this, both AMPK mRNA and activity are present in tissues sucn as liver, mammary gland, lung and brain, wnicr. are active lipid biosynthesis. However, tne nigh level of AMPK expression in skeletal and cardiac muscle, as well as therelatively low level of expression m adipose tissue, may indicate additional functions for tnis protein kinase. This is furtner supported oy our ocservatien that tne tissue-dependent expression cf tne rR A enclodmg ACC, wnicn is hown to pe a suostrate for AMFK in vivo, s inversely correlated witn expression of functional AMPK mRNA Although we have been unable to demonstrate appreciable AMPK activity m skeletal muscle, there are a number of possible substrates for the Kinase this tissue Glycogen synthase and phosphoryiase :mase pur fieα from rabbit skeletal muscle have both been snown to be pnospnorylated oy AMPK vitro An increased energy demand during - iscie contraction may lead to an increase in AMP Subsequent activation of AMPK could result in phosphorylation and mactivation of glycogen synthase and concomitant mobilisation of glycogen for the regeneration of ATP Rat skeletal (and cardiac) muscle appear to predominantly express a distinct isoform of acetyl-CoA carooxylase 280 rfith molecular mass of 280 kDa (ACC ) . It is not known whether 265 280 ACC and ACC arise from distinct genes, or from a common gene by alternative splicing. cDNA clones encoding the 265 kDa form, but not the 280 kDa form, have been isolated. The 280 kDa isoform has been proposed to play a role in the control of fatty acid oxidation in muscle Although there is good evidence that it is regulated by pnospnoryiation, to date a role for AMPK has not been demonstrated. 280 However, f ACC is phosphorylated and inactivated by AMPK, then activation of AMPK during increased muscle contraction would lead to a decrease in malonyl-CoA concentration. Malonyl-CoA is a potent inhibitor of carnitme palmitoyltranserase I, a key regulatory enzyme the transport of fatty acids into the mitochondria. Inhibition of 280 ACC by AMPK would therefore lead to an increase m the rate of fatty acid oxidation, and hence ATP production. Example 6 AMINO ACID SEQUENCE OF AMPK AND HOMOLOGY WITH OTHER PROTEIN KINASES A searcn of the Swiss-Prot database with the predicted protein sequence of AMPK reveals homology with the catalytic domain of other πembers of the protein kinase family. Interestingly, however, AMP snows a very hign degree of nomclogy (47% identity) with a protein kinase from Saccnaro yces cerevisiae termed SNFl, wnich s mvo veo n careen cataoolite oerepression (Celenza and Carlson 1986) The am o acid sequences of AMPK and SNFl protein kinases are compared in Figure 6 Sequence identity extends throughout the entire protein sequences, although outide of the catalytic domain (which corresponds to the N- erminal halves of the enzymes) the similarity is less extensive. The polyhistidme tract present at tne N-terminus cf SNFl s not present m AMPK. However, mutagenesis studies m wnicn 12 cf tne 13 consecutive histme residues were deleted have shown that this sequence is not necessary for SNFl function (Celenza and Carlson, 1989) . A cDNA clone encoding a putative protein kinase has recently oeen isolated from rye (RKIN1) , and the predicted ammo ac d sequence cf this clone shares extensive homology (46% identity) with SNFl (Alderson et al . , 1991) and AMPK (data not shown) . The predicted ammo acid sequence of AMPK contains a number of serme and threon e residues that lie withm good consensus sequences for phosphorylation by known serme/ hreonme protein kinases, In addition, AMPK contains several potential phosphorylation residues just N-termmal to the conserved Ala-Pro-Glu (APE) motif. Phosphorylation of residues in this region has been shown to be important for the regulation of other protein kinases (Sutherland et al. , 1993) . In Saccharomyces cerevisiae SNFl protein kmase activity is essential for release of genes from glucose repression. As already details we have obtained cDNA clones encoding an AMP-activated protein kinase (AMPK) that has been shown to be involved in the regulation of both fatty acid and cholesterol metabolism. The ammo ac d sequences of SNFl and AMPK are 46% identical. We investigated whether SNFl and AMPK shared any functional similarity in addition to their primary sequence homology. We used a synthetic peptide (the "SAMS" peptide whicn s based on the sequence surrounding one of the sites pnospnorylated within acetyl-CoA carboxyiase by AMPK as a suostrate m a onosphorylation assay in yeast extracts. We were able to detect kmase activity using this assay in w ld type yeast extracts which was immunoprecipitated by antibodies raised against either SNFl or SNF4. The activity of SNFl has previously been shown to be dependent on the association cf SNF4, whicn is though to be an activatory subunit of SNFl. It is noteworthy tnat we nave identified a 36 kDa polypeptide wn en co-purifies with rat AMPK by Western blotting using anticodies raised against SNF4 (data not snown) . We celieve that this polypeptide is likely to be involved in the regulation of mammalian AMPK in the same way that SNF4 regulates SNFl function m yeast. Protein kinase activity assayed oy the phosphorylation of the SAMS peptide was not detectable in a numoer of mutant yeast strains wnicn have previously been snown to be defective m SNFl function. These results confirm that the protein kmase activity we were measuring w th the SAMS peptide substrate was that of SNFl. Radiosequencmg of SAMS peptide (or its parent peptide, SSMS) phosphorylated by SNFl or AMPK revealed that the same serme residue, corresponding to serme-79 m rat acetyl-CoA carboxyiase, was labelled n either case idata not shown) . The apparent Km of SAMS peptide for SNFl (20μM) was similar to that of the AMPK (data not shown) . However, unlike AMPK we were unable to detect any significant activation of SnFl by AMP over a range of AMP concentrations (data not shown) . Since SNFl activity is known to be essential for release from glucose repression we looked at the actvitiy of SNFl isolated from wild type grown in derepressmg conditions (growth m 0.05% glucose) . We have found that the activity of SNFl increases significantly (3 fold) over time during growth under derepressmg conditions . This increase m activity was not due to a change in the amount of SNFl protein, as detected by Wastern blotting. The time course of SNFl activation precedes the previously reported time course of mvertase activation during derepression, consistent with the dea that SNFl protein k ase activity is necessary for activating mvertase gene expression. The increase in SNFl activity is due to a covalent modification since the effect survives purification by chromatography on DEAE-Sepharose. We therefore investigated the effect of dephosphorylation on SNFl activity Following incubation under conditions that would activate endogenous protein phsophatases present in the yeast extract the activity of SNFl was dramatically reduced. Furthermore, SNFl was inactivated by incubation w th the purified catalytic subunit of mammalian protein phosphatase 2A. These results indicate that, like AMPK, SNFl is itself regulated oy phospnorylation. Althougn our results do not discriminate between an autoonosphorylation reaction ano pnospnorylation and activation cy a distinct protein kinase, the similarities oetween SNFl and AMPK, forms part of a protein kinase cascade, and that SNFl is phospnorylated and activated during growth under derepressmg conditions. Since SNFl and AMPK snare functional homology we examined the activity of acetyl-CoA carcoxvlase, a substrate for AMPK m v vo, under derepressmg conditions yeast. We have found tnat the activity of acetyl-CoA carboxyiase isolated from yeast grown in 0.05% glucose (derepression) compared to yeast grown in repressing conditions (2% glucose) is decreased approximately 3-fold. The decrease m acetyl-CoA carboxyiase activity was due to phosphorylation and mactivation of the enzyme since the effect was observed after purification of acetyl-CoA carboxyiase to homogeneity by chromatography on avidin-Sepharose and could be reversed following dephosphorylation using the catalytic subunit of protein phosphatase 2A. The activity of acetyl-CoA carboxyiase assayed m crude extracts of snfl mutant yeast was approximately double that compared to wild type (repressed) . Acetyl-CoA carboxyiase purified from yeast is phospnorylated and inactivated by SNFl bound to anti-SNFl antibody protein A-Sepharose beads in vitro. The simplest interpretation of these results is that SNFl phosphorylates and inactivates acetyl-CoA carboxyiase in vivo. Yeast acetyl-CoA carboxyiase does not contain the homologous ammo acid sequence corresponding to the mactivatory AMPK onosphorylation site in tne rat enzyme (serme-79) although there are a number of potential phosphorylation sites withm yeast acetyl-CoA carboxyiase. Example 7 IMMUNOPRECIPITATION OF AMPK Antioodies raised against four different syntnetic peptides based on the deduced protein sequence of AMPK recognised a 63 kDa protein from rat liver by Western blotting (data not shown) . Incucaticn cf partially purified AMPK from rat liver fraction Carlmg et a . , 1989a) witn tne different antisera did not result any detectaoie less AMPK activity (data not snown 1 However, following precipitation of tne immune complex witn protein A-Sepnarose there was a loss in activity of AMPK in the supernatant fraction (Figure 7A) . Furthermore, with three of the four antisera. AMPK activity was detected in the immunoprecipitated complex (Figure "3 and data not snown) . 3y increasing tne amount cf antiserum added t was possible to increase the amound cf AMPK activity that could be precipitated from the supernatant (Figure 7B. AMPK activity remaining in the supernatant or present in the immune complex precipitate showed identical stimulation by AMP (approximately 3-fold, data not shown) . AMPK that had been metaoolically labelled by incubation of Sf9 cells infected with recomomant AMPK baculovirus with [ S]methion e was specifically immunoprecipitated from the cell lysate by the different anti-peptide antibodies (Fgire 7C) . No AMPK activity was detected in any of the immunoprecipitates from Sf9 cells which may be due to the absence of phosphorylation of AMPK m this system (unpublished results) . Example 8 PRODUCTION OF ANTI-AMPK ANTIBODIES Based on an antigemcity index (Al) of the cloned rat cDNA sequence (described by Jameson & Wolf, CABIOS, 1988, 4., 181-186) , four 15-mer peptides w th high Al values were synthesised. These were peptides based on the predicted ammo acid sequences of AMPK for residues 278-291 (PEDPSYDANVIDDE) , 361-374 (PGLKPHPERMPPLI) , 476-489 (WEQRSGSSTPQRS) , 496-509 (HRPRSSVDSSTAEN) . They were synthesised and coupled to bovine thyroglobulm (Sigma T1001) via a cystein residue added to the N-terminus of the peptide using M-maleimidocenzoyl-N-hydroxy succmimide ester (Pierce Ltd) (Green et al, Ceil, 1982, 2_8, 477) . Male New Zealand white rabbits (3 per conjugate) were immunised and bled as described previously (Forder et al, 1983, . 12., 323) . Specific antibody binding was demonstrated by cmdmg to respective peptides m a sandwich ELISA (Forder et al, preparation) Reaction Sequence Conjugates were subject to hydrolysis for 24 hours at 130°C in 6N/Phenol prior to amino acid analysis. The cystein residue in the peptide is converted to S-succinylcysteine K/tt, - L - Le»H i " 1 i v which is a small, fast runnning identifiable peak on the amino acid analyser. From the number of moles of succinylcysteine formed and knowing the amino acid composition of bovine thyroglobulin, it is possible to determine the molar incorporation of peptide per mol of thyroglobulin. In this instance, less than 10 moles peptide/mol thyroglobulin were not used to immunise animals e.g. residues 476-489 = 32, 361-374 = 18, 466-509 = 24, 278-291 = 38. Immunoprecipitation of AMPK Immunoprecipitation of AMPK was carried out by adding the indicated volume of serum to the kinase preparation followed by incubation at 4°C for 3-12 h. The volume of antiserum added was kept constant by the addition of preimmune serum were necessary. A 50% (w/v) slurry of protein A-Sepharose was added and the incubation continued for a further hour at 4°C. The mixture was centrifuged at 10,000 x g for 10 min. and the supernatant removed and assayed for AMPK activity where necessary. The pellet was washed with 3 x 100 μl cf buffer A containing 1% Triton X- 00 and resuspended n this ouffer for measuring AMPK activity or n SDS-sample ouffer for electrcpnoretic analysis. METHODS AND MATERIALS Purification of AMPK AMPK was purified from rat liver as far as the Superose-12 gel filtration step, exactly as described previously (Carlmg et al. , Eur.J.Biochem. , 1989, 186, 129-136) . Approximately 50 μg of purified AMPK polypeptide (yield determined from Coomassie stained polyacrylamide gels and comparison with known amounts of standards) was obtained from 150 g tissue (10 rats) and used to derive am o acid sequence information. The partially purified kinase preparation was resolved by SDS-PAGE on 10% polyacrylamide gels (Lammlli, Nature, 1970, 227, 680-685) . AMPK assay AMPK activity was assayed in vitro by the phosphorylation of a syntnetic peptide substrate (HMRSAMSGLHLVKRR, the 'SAMS' peptide) as described by Davies et al. (Eur.J.Biochem, 1989, 186, 123-128) . Unless indicated otherwise assays were performed at 30° for 30 mms in the presence of 200 μM AMP using the SAMS peptide at a concentration of 200 uM. Isolation and amino ac d sequencing of AMPK peptides The 63 kDa polypeptide identified as AMPK was excised from SDS-polyacrylamide gels following electrophoresis and cleaved either enzymatically (S. aureus V8 protease or endoprotemase lys-C) or cne ically 'cyanogen cromide) Proteolytic digestion was performed during electrophoresis oy tne method of Cleveland et al . .Chem. , 1977, 252 , 1102-1106) Following electropnoresis tne peptides were transferred to FVDF memcrane, visualised by staining with Coomassie and subjected to automated ammo acid sequence analysis. Chemical cleavage was performed by overnight incubation of the gel slice withcyanogen oromice n 90% formic acid at room temperature. The supernatant was removed and dried in a Speed-Vac. The resiαue was wasneα twice with water, dried and resuspended in SDS-gel loading buffer. Peptides were resolved by SDS-PAGE using a tricine buffer system (Schagger and von Jagow, Anal .Biochem. , 1987, 166, 368-379) and transferred to PVDF membrane for sequencing. Sequencing was carried out on an Applied Biosvstems A431 automated sequencer. Amplification of AMPK cDNA Degenerate oligonucleotides were synthesised corresponding to the N- and C-terminal sequences of peptide ELI. These oligonucleotides were then used to obtain the cDNA sequence that encodes ELI. Rat liver mRNA (1 μg) was reversed transcribed with a mixture of oligo dT and random hexamer primers (BRL Pre-Amplification kit) . An aliquot of th s reaction was diluted (1:100) and used in a polymerase chain reaction with the following primers: 5' NNNCCNGA(A/G)AA(C/T)GTN(C/T)TN(A/C/T) (C/T)NGA 3' (sense) and 5' CAT(A/G)TTN(C/G) (A/T)NA(A/G)NCC (A/G)AA(A/G)TCNGC 3' (antisense) for 40 cycles of 94°C, 2 mm: 55°C, 2 mms : 72°C, 2 mm. The products of this reaction were diluted (1:200) and used for a second round of amplification (same conditions as before with "nested" primers: 5' CCNGA(A/G)AA(C/T)GTN(C/T)TN(A/C/T) (C/T)NGA(C/T) G 3' (sense) and 5' (A/G)TTN(C/G) (A/T)NA(A/G)NCC (A/G)AA(A/G)TCNGC(A/G) TC 3' (antisense) . The products of the reaction were analysed by eletrcpnoresis on a low-metltmg point agarose gel and a band of the expected size (63 nt) identified by ethidium bromide staining. The DNA was purified from the gel, blunt-ended with T4-polymerase and ligated into Bluescπpt. Plasmids containing an insert weRe identified by restriction digestion and sequenced by dideoxy chain termination (Sanger et al . , 19"" 7 using Sequenase 2.0 and vector primers Isolation of AMPK cDNA clones Standard molecular biology techniques were used (Sa orooK et al., 1989) . A unique oligonucieotide (5' GACGCCCAGATGAATGCTAAG 3') cased on the sequence of the PCR product was used to probe a rat liver cDNA library iamodaZAP II, Stratagene) Hybridisation conditions 9 6 were: 30 ng prooe (-10 c.p.m/ug; ID c.p.m/ml) in 5 x SSPE, 100 ug/ l sonicated and denatured salmon sperm DNA, 2 x Denharts, 0.1% SDS at 50°C for 4 hours. Filters were washed with 5 x SSC, 0.1% SDS at room temperature for 2 hours, followed by 5 x SSC, 0.1% SDS at 50°C for 30 mm and autoradiography for 18 hours at -70°C with intensifying screens. A total of 1.5 x 10 plaques were screened and eleven hybridising clones were isolated. Plasmids were recovered by m vivo excision from the phage (Short et al. , Nucl.Acid.Res. , 1988, 16., 7583-7600) . Analysis of the inserts by restriction endonuclease mapping revealed that the clones could be divided into two groups : clones PK-1 (8 clones) cont aned an insert of -2.6 kb, whilst 1 — 8 clones PK-2 (3 clones) had an insert of -2.7 kb. The inserts from both sets of clones were sequenced on both strands with either vector or AMPK cDNA specific primers. Preparation of pBS-APK In order to obtain a cDNA clone containing the entire coding sequence of AMPK, rat liver cDNA was amplified using primers based on clones PK-1 and PK-2 (foward prιmer-5' GCCGAACATGGCTGAGAAG 3' nt 1-19: reverse primer-5'TCTTAGCATTCATCTGGGC 3'; antisense nt 452-470) . The larger of the products was purfied by electrophoresis on a low-meltmg point agarose gel, recovered using Magic DNA clean-up resm (Promega) , cloned into Bluescπpt and sequenced to confirm the orientation and identity of the insert. This plasmid was then digested with NotI and Bglll to release a fragment of -185 bp. The Notl-Bglll fragment was purified and ligated into PK-1 that had been digested w th NotI and Bglll. The resulting clone (pBS-APK) was isolated and sequenced across the Notl-Bglll region to verify its integrity. RNA analysis Total RNA was isolated from various rat tissues (Chomczynski and Saacni, 13871. RNA l ~ ) was reverse transcribed with AMV-RT at 42°C for 30 mm. using random hexamers . An aliquot of this reaction was used in PCR with various AMPK specifc primers under the following conditions; initial denaturing step cf 94°C for 3 mm. followed by 30 cycles of 94°C, 1 mm; 55°C, 1 mm; "2°, 1 mm. Products were analysed by electrophoresis on agarose gels. Rat multiple tissue Nortnern (Clontech) was probed with either a random primed (Femcerg and Volgelstem, Anal. Biochem. , 1983, 132, 6-13) 1.9 kb EcoRI fragment from pBS-APK, or a rat GAPDH fragment, according to the manufacturers instructions. The blot was washed with 2 x SSC, 0.5% SDS at room temperature for 1 hour, followed by 0.2 x SSC. 0.5% ΞCS at 65°C for 2 x 20 mm. Southern blotting Rat genomic DNA (10 μg) was digested overnight at 37°C with various restriction endonucleases. DNA was electrophoresed on a 1% agarose gel and tranferred to a nylon membrane (GT membrane, Bio-Rad) . The membrane was probed with the entire insert from clone PK 1 using the following hybridisation conditions: 5 x SSPE, 100 μg/ml sonicated and denatured salmon sperm DNA, 2 x Denhardts, 0.1% SDS, 50% formamide at 42°C for 16 hours. Filters were washed with 2 x SSC, 0.5% SDS at room temperature for 1 hour followed by 0.1 x SSC, 0.5% SDS at 65°C for 2 x 20 mm. Expression of AMPK using the baculovirus system The 1.9 kb EcoRI fragment pBS-APK was inserted into the baculovirus transfer vector, pVL1392 (AMS Biotechnology) . The correct orientation of the insert was determined by digestion with BamHI yielding the recombmant vector, VL-APK. Approximately 2 μg pVL-AMP (purified by caesium chloride density gradient centrifugation) and 1 μg BaculoGold linearised baculovirus DNA (AMS Biotechnology) was co-transfected into Sf9 cells using calcium phosphate preciptation. Recomoinant viral particles were amplified through two rounds of infection, before infecting a large scale preparation of insect cells 1C " cells: m.c. . 10) . Cells were grown at 27°C and were harvested just trier to lysis (approximately " 2 r.ours) . After centrifugation ceils were iysed by resuspension 53 mM Tπs-KCl [pH 7.5] , 50 mM NaF, £ mM Napyrophosphate, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10% (v/v) glycercl (buffer A) containing 1% Triton-X 100. The resulting material was centrifuged at 10,000 x g for 15 min. For metabolic labelling Sf9 cells were incubated with [ ~S] methionme for 1 hour, 72 hours post-infection, and lysed as above. The supernatant fraction was used for immunopreciptation cf recombinant AMPK. Table 1 Full length cDNA sequence of the rat liver AMP-PK [translation initiation codon (Methionine) is underlined! . CCCGGGGAGG CCGCGCGCCG AACATGGCTG AGAAGCAGAA GCACGACGGG 51 CGTGTGAAGA TCGGACACTA CGTGCTGGGG GACACCCTGG GCGTCGGCAC 101 CTTCGGCAAA GTGAAGATTG GAGAACATCA ATTGACAGGC CATAAAGTGG 151 CAGTTAAGAT CTTAAATAGA CAGAAGATTC GCAGTTTAGA TGTTGTTGGA :01 AAAATAAAAC GAGAAATTCA AAATCTTAAA CTCTTTCGTC ATCCTCATAT 151 TATCAAACTC TACCAAGTGA TCAGCACTCC AACAGACTTT TTTATGGTAA 301 TGGAATATGT GTCTGGAGGT GAATTGTTCG ACTACATCTG TAAACACGGG 351 AGGGTTGAAG AGGTGGAAGC TCGCCGGCTC TTCCAGCAGA TTCTGTCTGC 401 CGTGGACTAC TGTCACAGGC ACATGGTTGT CCACAGGGAC CTGAAGCCAG 451 AGAACGTGTT GCTGGACGCC CAGATGAATG CTAAGATAGC TGACTTCGGA :TCTCTAATA TGATGTCAGA TGGTG ATTT CTACGAACTA GCTGTGGATC GCCAAATTAT GCAGCACCGG AGGTCATCTC AGGAAGGCTG TATGCGGGTC 601 CTGAGGTTGA TATCTGGAGC TGTGGTGTTA TCCTGTATGC CCTTCTCTG 1 ] GGCACCCTCC CGTTCGACGA TGAGCACGTG CCTACGCTCT TTAAGAAGAT CCGAGGGGGT GTGTTCTACA TCCCGGAGTA TCTCAACCGT TCTATTGCCA 751 CTCTGCTGAT GCACATGCTG CAGGTGGACC CCTTGAAGCG AGCAACTATC 8 01 AAAGACATAC GAGAGCATGA ATGGTTTAAA CAGGATTTGC CCAGTTACCT 851 CTTTCCTGAA GACCCCTCCT ATGATGCTAA CGTCATTGAT GATGAGGCTG 901 TGAAAGAAGT ATGTGAAAAA TTTGAGTGTA CAGAATCAGA AGTGATGAAC 951 AGTTTATACA GTGGTGACCC TCAAGACCAG CTCGCAGTGG CTTATCATCT 1001 CATCATTGAC AATCGGAGAA TAATGAACCA AGCCAGTGAG TTCTACCTCG 1051 CCTCCAGTCC TCCAACGGGT TCCTTCATGG ACGATATGGC CATGCACATT 1101 CCCCCCGGCC TGAAACCACA TCCTGAAAGG ATGCCACCTC TCATAGCAGA 1151 CAGCCCCAAA GCACGCTGTC CACTGGATGC ACTCAACACA ACTAAGCCCA : 01 AATCTTTAGC TGTGAAAAAA GCCAAGTGGC ACCTTGGGAT CCGAAGCCAG • 1 AGCAAACCAT ACGACATTAT GGCGGAGGTG TACCGAGCTA TGAAGCAGCT GGACTTTGAA TGGAAGGTAG TGAATGCATA CCATCTTCGA GTAAGAAGAA .3 51 AAAACCCAGT GACTGGCAAT TACGTGAAAA TGAGCTTACA GCTTTACCTG 3TTGACAATC GGAGCTATCT TCT GACTTT AAAAGCATCG ATGATGAGGT ■iCACC 1501 CCGGCCTCCA CAGACCTCGG TCAAGTGTCG ATTCCAGCAC AGCCGAGAAC ΓTCACTGT CTGGCTCTCT CACTGGTTCT TTGACTGGCA GAACTTTGTC .601 CTCCGCTTCC CCGCGCCTGG GCAGTCATAC CATGGATTTT TTTGAAATGT 1651 GCGCCAGTCT TATCACTGCT TTAGCCCGTT GATAACCCAC CACCGGTCTC .701 TGTCTTTCTG TTACCGCACT GTGAAATCAC ATACACTCTT CAAATTATTA .751 CCGCACTCTC GGGTACCACA GGCTCTGCAA TAGAAGTTAT GTGAACATTC 1801 CCAGGTGACA TGCAGTGCTG CTGGAAACAC AGAAATCTGG CCTTCTGTTT 1851 ACTTTTAGAA CTCTGTAACT CTGCTGTGCC TATGATAGGT ATCAATAGCT 1901 AGGAACGGCT GAGTGCTGGT GAAGCTTGTT AACTTACACC CGTGAATTC? 1951 CTACACATGG TGAGCACACC TCACTGATGA ACCCGCTGAT CTCGGGGTGG 2001 TTCGGTGGGA CCGCCTTCCT TCACGTTTAG TTCATGTAAA TCCTGTTTGC 2051 CTCCTAAATT TCCATAGGTG TCAGGCTGTC TAGGCACTCT TGGACAAGAA 2101 GATTCAGAAA TAGAGTAACT GTCAGTGAAA TATTATTTAA ATGTAGAAAT 2151 CCGAAATCCT GTCCCCTTAA ATATCAGAAA CCAAAAGTCT TTTTTAATAC TTTCTGCAAA TACTGCCTAG TATTAGCCAT ACAGACTGTG TTTCTGATAA 2251 TAGGAGCCCA GTCTCCTAGC TTCCTGACAT TTGTGCAAGG CCCCTAGTTA AATCCCACTA CCACAAAGCA ATATAAAACC ATGCAAAGTT AGTGCATAGT TAAAGGAAAC AGGCAGATAC CAACAGTCTC TTAAAAGGAA ATCTATTCTT 2401 TGATCTCATT TGTGTTTATG AGACTGGGTA GCTGGGGGCT TGGGGGCGGG :451 CTGGGAGTCA CAGCCCTTGG GCATCTTTGC TAATCCACTT GAACTTTGT1 :501 ATTGATGCCG AGTCCTCTCT CTTCCTCAGT AGCCACTGTC TTTGCTCATT 2551 GCTTTTCCCT TTTTTAACTC CTTTAGATCA AAACCTTGCT TTGGCCATAG 2601 GGTCTTTAAA TACTTTCAAA GCTTGATCTG CTGTGACCTT CACTGTTGAA 2651 CCTGATTGGA CAGGGAAGCC TTAAATATTT AAAAGTATAT TCTCCTGGAA 2701 TAACTACATG TGTTGTTTAC ATATATATAC ATATGTATTG ACACATCAGT 2751 GCTTTTAATC G Table 2 Tabled Peptide sequence generated from the 63kd protein bv proteolytic and chemical cleavage PEPTIDE (SIZE) SEQUENCE PK0 (17K/18K) XLXPENVL(T/L)DA(Q P)MNAXDADFG(L)SNM BT2 (7K) EYV(Y)G(K L)(E)LF(A)YIXKXGXXXXV BT3 (12K) LQV(D)PLK(R)ATIK(D)IXE(E)E(W)FKQ BT4 (8K) PPLIADXPKARXP D(A)LN(η(T)K BT1 (7K 14K) KQLDFE(W/S)(K G)KVVNAY(H)L(R)V(R)XKXN X= UNCERTAIN ()= MOST LIKELY Enzyrnβg that do not cut* Λαtll Λccl Aflll Apal ApoLI Λβcl Λeol Avrll Bool Bcgl Bcgl B ll nzifnea exc uded! HlnCuts: 1 HαxCuts: 350000 Ntt£ Table 4 Amino Acid Sequence Information Obtained from Peptides Derived from AMPK Method of Cleavage Peptide Amino Acid Sequence Comments Comments Endoproteinase lys C ELI (K)PENVLTDAQMNAX(D)ADFGLSNM homology to protein homology to protein kinase L KI subdomains VIb-VII subdomains VIb-VII /T)DAQMNAX(D)ADFGLSNM sequence derived from L K I peptide ELI Cyanogen bromide CB1 (M)EYVSGGELFDYICKHGRVEE homology to protein kinase homology to protein kinase subdomain V subdomain V Table 4 (continued) CO Method of Cleavage Peptide Amino Acid Sequence Comments Comments CD Cyanogen bromide CB2 (M) PPLIADSPKARCPLDALNTTK CΛ c Cyanogen bromide CB3 (M) KQLDFEWKWNAYHLRVRRK Residues in brackets are either tentative assignments or assumptions based on the method of cleavage. Residues marked X could not be identified by amino acid sequencing. The amino acid sequences of all the peptides were found in the cDNA clone (Figure 3A) , except for the differences indicated below the peptide sequence. Table 5 1 GATGGCTGAG AAGCAGAAGC ACGACGGGCG GGTGAAGATC GGACACTACG 51 TGCTGGGCGA CACGCTGGGC GTCGGCACCT TCGGCAAAGT GAAGATTGGA 101 GAACATCAAT TAACAGGCCA TAAAGTGGCA GTTAAAATCT TAAATAGACA 151 GAAGATTCGC AGTTTAGATG TTGTTGGAAA AATAAAACGA GAAATTCAAA 201 ATCTAAAACT CTTTCGTCAT CCTCATATTA TCAAACTATA CCAGGTGATC 251 AGCACTCCAA CAGATTTTTT TATGGTAATG GAATATGTGT CTGGAGGTGA 301 ATTATTTGAC TACATCTGTA AGCATGGACG GGTTGAAGAG ATGGAAGCCA 351 GGCGGCTCTT TCAGCAGATT CTGTCTGCTG TGGATTACTG TCATAGGCAT 401 ATGGTTGTTC ATCGAGACCT GAAACCAGAG AATGTCCTGT TGGATGCACA 451 CATGAATGCC AAGATAGCCG ATTTCGGATT ATCTAATATG ATGTCAGATG 501 GTGAATTTCT GAGAACTAGT TGCGGATCTC CAAATTATGC AGCACCTGAA 551 GTCATCTCAG GCAGATTGTA TGCAGGTCCT GAAGTTGATA TCTGGAGCTG 601 TGGTGTTATC TTGTATGCTC TTCTTTGTGG CACCCTCCCA TTTGATGATG 651 AGCATGTACC TACGTTATTT AAGAAGATCC GAGGGGGTGT CTTTTATATC 701 CCAGAATATC TCAATCGTTC TGTCGCCACT CTCCTGATGC ATATGCTGCA 751 GGTTGACCCA CTGAAACGAG CAACTATCAA AGACATAAGA GAGCATGAAT 801 GGTTTAAACA AGATTTGCCC AGTTACTTAT TTCCTGAAGA CCCTTCCTAT 851 GATGCTAACG TCATTGATGA TGAGGCTGTG AAAGAAGTGT GTGAAAAATT 901 TGAATGTACA GAATCAGAAG TAATGAACAG TTTATATAGT GGTGACCCTC 951 AAGACCAGCT TGCAGTGGCT TATCATCTTA TCATTGACAA TCGGAGAATA 1001 ATGAACCAAG CCAGTGAGTT CTATCTCGCC TCTAGTCCTC CATCTGGTTC 1051 TTTTATGGAT GATAGTGCCA TGCATATTCC CCCAGGCCTG AAACCTCATC 11C1 CAGAAAGGAT GCCACCTCTT ATAGCAGACA GCCCCAAAGC AAGATGTCCA 1151 TTGGATGCAC T3AATACGAC AAGCCCAAA TCTTTAGCT3 TGAAAAAAGC 1 01 CAAGTGGCAT CTTGGAATCC GAAGTCAGAG CAAACCGTAT GACATTATGG Table 5 (continued) 1251 CTGAAGTTTA CCGAGCTATG AAGCAGCTGG ATTTTGAATG GAAGGTAGTG 1301 AATC-CATACC ATCTTCGTGT AAGAAGAAAA AATCCAGTGA CTGGCAATTA 1351 CGTGAAAATG AGCTTACAAC TTTACCTGGT TGATAACAGG AGCTATCTTT 1401 TGGACTTTAA AAGCATTGAT GATGAAGTAG TGGAGCAGAG ATCTGGTTCC 1451 TCAACACCTC AGCGTTCCTG TTCTGCTGCT GGCTTACACA GACCAAGATC 1501 AAGTTTTGAT TCCACAACTG CAGAGAGCCA TTCACTTTCT GGCTCTCTCA 1551 CTGGCTCTTT GACCGGAAGC ACATTGTCTT CAGTTTCACC TCGCCTGGGC 1601 AGTCACACCA TGGATTTTTT TGAAATGTGT GCCAGTCTGA TTACTACTTT 1651 AGCCCGTTGA TCTGTCTCTA GTTTCTTTCT GTTATTGCAC TATGAAAATC 1701 AGTTATATTC TTTAAATTTT TATCTTACTT TTGGATAATA TCCATCCAGC 1751 TTGGACTTAA CCAGGCTGAA CTTGCTCAAA AGG Table 6 NUCLEOTIDE SEQUENCE COMPARISON BETWEEN THE FULL-LENGTH cDNA SΞOUENCΞS OF CLONED RAT AND HUMAN AMP KINASES. THE SEQUENCE COMPARISON REVEALS AN IDENTITY OF 89.78% 2 ATGGCTGAGAAGCAGAAGCACGACGGGCGGGTGAAGATCGGACACTACGT 51 24 ATGGCTGAGAAGCAGAAGCACGACGGGCGTGTGAAGATCGGACACTACGT 73 52 GCTGGGCGACACGCTGGGCGTCGGCACCTTCGGCAAAGTGAAGATTGGAG 101 .MM! MM! IMMMMMMMMMMMMMMMMMM 74 GCTGGGGGACACCCTGGGCGTCGGCACCTTCGGCAAAGTGAAGATTGGAG 123 102 AACATCAATTAACAGGCCATAAAGTGGCAGTTAAAATCTTAAATAGACAG 151 MIIMMM MIMMMMMMMMIMI 111111111111111 124 AACATCAATTGACAGGCCATAAAGTGGCAGTTAAGATCTTAAATAGACAG 173 152 AAGATTCGCAGTTTAGATGTTGTTGGAAAAATAAAACGAGAAATTCAAAA 201 174 AAGATTCGCAGTTTAGATGTTGTTGGAAAAATAAAACGAGAAATTCAAAA 223 202 TCTAAAACTCTTTCGTCATCCTCATATTATCAAACTATACCAGGTGATCA 251 M! MMMMIMMMMMMMMMIMM MMI MMMI 224 TCTTAAACTCTTTCGTCATCCTCATATTATCAAACTCTACCAAGTGATCA 273 252 GCACTCCAACAGATTTTTTTATGGTAATGGAATATGTGTCTGGAGGTGAA 301 MMMMMMI MMMMMMMMMMMMMMMMMM 274 GCACTCCAACAGACTTTTTTATGGTAATGGAATATGTGTCTGGAGGTGAA 323 302 TTATTTGACTACATCTGTAAGCATGGACGGGTTGAAGAGATGGAAGCCAG 351 324 TTGTTCGACTACATCTGTAAACACGGGAGGGTTGAAGAGGTGGAAGCTCG 373 352 GCGGCTCTTTCAGCAGATTCTGTCTGCTGTGGATTACTGTCATAGGCATA 401 MMMM MMMMMMMM! !!l|t MiMMI MM! ! 374 CCGGCTCTTCCAGCAGATTCTGTCTGCCGTGGACTACTGTCACAGGCACA 423 402 7GGTTGTTCATCGAGACCTGAAACCAGAGAATGTCCTGTTGGATGCACAC 451 MMM M i MMMM MMMM M M MM M M 424 TGGTTGTCCACAGGGACCTGAAGCCAGAGAACGTGTTGCTGGACGCCCAG 473 452 ATGAATGCCAAGATAGCCGATTTCGGATTATCTAATATGATGTCAGATGG 501 MIMMI MMMM II MMM I II II I II I II 11 II 111 II I 474 ATGAATGCTAAGATAGCTGACTTCGGACTCTCTAATATGATGTCAGATGG 523 502 TGAATTTCTGAGAACTAGTTGCGGATCTCCAAATTATGCAGCACCTGAAG 551 ' : ' : , : ! I MM : ' ; M ■ ! : M : j ι j I i I II 11 : ' ' SSm m CATCTCAGGCAGATTGTATGCAGGTCC GAAGTTGATATCTGGΛGCT 602. MMM! M MMIM MIMMI MMMMMMMM!! 574 TCATCTCAGGAAGGCTGTATGCGGGTCCTGAGGTTGATATCTGGAGCTGT 623 Table 6 (continued) MMMM! Mill! 1 MM! I I M I I I ; 11 j i I ! M M lllli 624 GGTGTTATCCTGTATGCCCTTCTCTGTGGCACCCTCCCGTTCGACGATGA 673 652 GCATGTACCTACGTTATTTAAGAAGATCCGAGGGGGTGTCTTTTATATCC 701 Ml M MMM I ! M I 1111111 M 11111 M M I II II MM 574 GCACGTGCCTACGCTCTTTAAGAAGATCCGAGGGGGTGTGTTCTACATCC 723 702 CAGAATATCTCAATCGTTCTGTCGCCACTCTCCTGATGCATATGCTGCAG 751 ; M MM MM MM M I MM MM MMMM M IMM I! 724 CGGAGTATCTCAACCGTTCTATTGCCACTCTGCTGATGCACATGCTGCAG 773 752 GTTGACCCACTGAAACGAGCAACTATCAAAGACATAAGAGAGCATGAATG 801 II Mill MM 11111111111111111111! MMMMIMM 774 GTGGACCCCTTGAAGCGAGCAACTATCAAAGACATACGAGAGCATGAATG 823 802 GTTTAAACAAGATTTGCCCAGTTACTTATTTCCTGAAGACCCTTCCTATG 851 824 GTTTAAACAGGATTTGCCCAGTTACCTCTTTCCTGAAGACCCCTCCTATG 873 852 ATGCTAACGTCATTGATGATGAGGCTGTGAAAGAAGTGTGTGAAAAATTT 901 MMMIMMMIMIMMMMIMIMMMM MMMMIMI 874 ATGCTAACGTCATTGATGATGAGGCTGTGAAAGAAGTATGTGAAAAATTT 923 902 GAATGTACAGAATCAGAAGTAATGAACAGTTTATATAGTGGTGACCCTCA 951 M M MMMMMMM! ! 1111 M I I 1111 I !! 11111111 ! 11 I 924 GAGTGTACAGAATCAGAAGTGATGAACAGTTTATACAGTGGTGACCCTCA 973 952 AGACCAGCTTGCAGTGGCTTATCATCTTATCATTGACAATCGGAGAATAA 1001 MMIMMMIMM! II I II II II I II 111 II 11111 974 AGACCAGCTCGCAGTGGCTTATCATCTCATCATTGACAATCGGAGAATAA 1023 1002 TGAACCAAGCCAGTGAGTTCTATCTCGCCTCTAGTCCTCCATCTGGTTCT 1051 MMMMMMIMMIMM MIMMI 11111 ! I M I Mill 1024 TGAACCAAGCCAGTGAGTTCTACCTCGCCTCCAGTCCTCCAACGGGTTCC 1073 1052 TTTATGGATGATAGTGCCATGCATATTCCCCCAGGCCTGAAACCTCATCC 1101 M M I M M M MMMM MM M M MMMIIM! M il l 1074 TTCATGGACGATATGGCCATGCACATTCCCCCCGGCCTGAAACCACATCC 1123 1102 AGAAAGGATGCCACCTCTTATAGCAGACAGCCCCAAAGCAAGATGTCCAT 1151 MIMMIMMIIM! ! I II II II 11 II I! 11 II II I I MMM 1124 TGAAAGGATGCCACCTCTCATAGCAGACAGCCCCAAAGCACGCTGTCCAC 1173 1152 TGGATGCACTGAATACGACTAAGCCCAAATCTTTAGCTGTGAAAAAAGCC 1201 1174 TGGATGCACTCAACACAACTAAGCCCAAATCTTTAGCTGTGAAAAAAGCC 1223 Table 6 (continued) 1202 AAGTGGCATCTTGGAATCCGAAGTCAGAGCAAACCGTATGACATTATGGC 1251 i M M M i I ! I I i i I I i I i i i i ! M I I M I I M M i 1224 AAGTGGCACCTTGGGATCCGAAGCCAGAGCAAACCATACGACATTATGGC 1273 125 TGAAGTTTACCGAGCTATGAAGCAGCTGGATTTTGAATGGAAGGTAGTGA 1301 II II MMMMMMMMMIMM MMMMMMMIMM 1274 GGAGGTGTACCGAGCTATGAAGCAGCTGGACTTTGAATGGAAGGTAGTGA 1323 1302 ATGCATACCATCTTCGTGTAAGAAGAAAAAATCCAGTGACTGGCAATTAC 1351 M M M M I M I M M M M M M M M M M M M M I M M I M M 1324 ATGCATACCATCTTCGAGTAAGAAGAAAAAACCCAGTGACTGGCAATTAC 1373 1352 GTGAAAATGAGCTTACAACTTTACCTGGTTGATAACAGGAGCTATCTTTT 1401 MMMMMMIMM MMMMM MM I! IMIMIMM I 1374 GTGAAAATGAGCTTACAGCTTTACCTGGTTGACAATCGGAGCTATCTTCT 1423 1402 GGACTTTAAAAGCATTGATGATGAAGTAGTGGAGCAGAGATCTGGTTCCT 1451 11111111111111 MMMM II IMIIMIMI MIMMI I 1424 AGACTTTAAAAGCATCGATGATGAGGTGGTGGAGCAGAGGTCTGGTTCTT 1473 1452 CAACACCTCAGCGTTCCTGTTCTGCTGCTGGCTTACACAGACCAAGATCA 1501 MIM M MM M MMM M M M M M l I MIMMI I I II 1474 CAACACCTCAGCGCTCCTGTTCTGCTGCCGGCCTCCACAGACCTCGGTCA 1523 1502 AGTTTTGATTCCACAACTGCAGAGAGCCATTCACTTTCTGGCTCTCTCAC 1551 ill ! MMM! I! II MM MIMMI! 11111 M 1111 I I I 1524 AGTGTCGATTCCAGCACAGCCGAGAACCATTCACTGTCTGGCTCTCTCAC 1573 1552 TGGCTCTTTGACCGGAAGCACATTGTCTTCAGTTTCACCTCGCCTGGGCA 1601 Ml IIIMIM II II II Mill II I III II MIIMMII 1574 TGGTTCTTTGACTGGCAGAACTTTGTCCTCCGCTTCCCCGCGCCTGGGCA 1623 1602 GTCACACCATGGATTTTTTTGAAATGTGTGCCAGTCTGATTACTACTTTA 1651 MM i I i I I II M 11 M 11 ! I I l I I II MMMM I! Ml MM! 1624 GTCATACCATGGATTTTTTTGAAATGTGCGCCAGTCTTATCACTGCTTTA 1673 1652 GCCCGTTGAT CTGTCTCTAGTTTCTTTCTGTTATTGCACT 1691 MM MM! MM 1 ' MMIMMM 1674 GCCCGTTGATAACCCACCACCGGTCTC...TGTCTTTCTGTTACCGCACT 1720 1692 ATGAAAATCAGTTATATTCTTTAAATTTTTATCTTACTTTTGGATA 1737 I I MMM M I MM Mi l l Ml ι III I II M 17 1 GTG . AAATCACATACACTCTTCAAATTATTACCGCACTCTCGGGTA 1765 SEQUENCE LISTING GENERAL INFORMATION (i) APPLICANT: ZENΞCA Limited (ii) TITLE OF INVENTION: NUCLEIC ACID (iii) NUMBER OF SEQUENCES: 39 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Group Patents Services Department (B) STREET: Bessemer Road (C) CITY: Welwyn Garden City (D) STATE: Hertfordshire (E) COUNTRY: United Kingdom (F) ZIP: GB-AL7 1HD (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb Storage (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS 3.20 (D) SOFTWARE: ASCII from WPS-PLUS (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER (B) FILING DATE: ! • :::' PRIOR APPLICATION DATA: ( A) APPLICATION NO: 9310489.1 (B) FILING DATE: 21-May-1993 (A) APPLICATION NO: 9318010.7 (B) FILING DATE: 31-Aug-1993 19 - (2) INFORMATION FOR SEQ ID NO:1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: peptide (C) STRANDEDNESS : single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : X L X P E N V L (T/L) D A (Q/P) M N A X D A D F G (L) S N M 25 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear ixi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : ;L) R P L Y D I P Y H F E A R (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : A T (T/L) (F/E) T X E L V L Q R E V E V E V E S M D K A G N F 27 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: ΞΞQ ID NO:4 : NNN CCN GA(A/G) AA(T/C) GTN (C/T)TN (C/T/A) (C/T)N GA (2) INFORMATION FOR SEQ ID NO: 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids ' (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : X P E N V L T/L D 8 (2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear i) SEQUENCE DESCRIPTION: SEQ ID NO: 6 : CCN GA(A/G) AA(T/C) GTN (C/T)TN (C/T/A) (C/T)N GA(T/C) G 21 ( ) INFORMATION FOR SEQ ID NO:7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 ammo acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: P Ξ N V L T/L D A (2) INFORMATION FOR SEQ ID NO: 8 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (Xl) SEQUENCE DESCRIPTION: SΞQ ID NO: 8 : CAT Z- A)TT N!G C) (A/T) NA(G/A) NCC (G/A)AA (A/G) TC NGC '2) INFORMATION FOR SEQ ID NO:9 : (i) SEQUENCE CHARACTERISTICS: (A) LΞNGTH: 8 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : M N S L G F D A 8 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: (G/A)TT N(G/C) (A/T) NA(G/A) NCC (G/A)AA (A/G) TC NGC (G/A) TC (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LΞNGTH: 8 amino acids (B) TYPE: peptide (C) STRANDEDNESS : single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11; N S L G F D A D (2) INFORMATION FOR SEQ ID NO:12 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GCCCAGATGA ATGCTAAG 18 (2) INFORMATION FOR SΞQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: A P M N A K 6 (2) INFORMATION FOR SEQ ID NO:14 : (i) SEQUENCΞ CHARACTERISTICS: (A) LENGTH: 18 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear > x SEQUENCE DESCRIPTION: SEQ ID NO: 14 : GACGCCCAGA TGAATGCTAAG 18 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCCGAACATG GCTGAGAAG 19 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TCTTAGCATT CATCTGGGC 19 INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LΞNGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAAGATCGGA CACTACG 17 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ;xi) SEQUENCE DESCRIPTION: SEQ ID NO:l£ CCTCCAGACA CATATTCC 18 INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: P E D P S Y D A N V I D D E 14 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: P G L K P H P Ξ R M P P L 14 (2) INFORMATION FOR SΞQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: V V E Q R S G S S T P Q R S 14 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCΞ CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear ;>:i) SEQUENCE DESCRIPTION: SEQ ID NO:22: H R P R S S V D S Ξ T A Ξ N 13 ,2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: H M R S A M S G L H L V K R R 15 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2761 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .xi) SEQUENCΞ DESCRIPTION: SEQ ID NO:24: CCCGGGGAGG CCGCGCGCCG AACATGGCTG AGAAGCAGAA GCACGACGGG CGTGTGAAGA 60 TCGGACACTA CGTGCTGGGG GACACCCTGG GCGTCGGCAC CTTCGGCAAA GTGAAGATTG 120 GAGAACATCA ATTGACAGGC CATAAAGTGG CAGTTAAGAT CTTAAATAGA CAGAAGATTC 180 GCAGTTTAGA TGTTGTTGGA AAAATAAAAC GAGAAATTCA AAATCTTAAA CTCTTTCGTC 240 TAT TATCAAACTC TACCAAGTGA TCAGCACTCC AACAGACTTT TTTATGGTAA 3 00 TGGAATATGT GTCTGGAGGT GAATTGTTCG ACTACATCTG TAAACACGGG AGGGTTGAAG 360 ^GGTGGAAGC TCGCCGGCTC TTCCAGCAGA TTCTGTCTGC CGTGGACTAC TGTCACAGGC 420 :ATGGTTGT CCACAGGGAC CTGAAGCCAG AGAACGTGTT GCTGGACGCC CAGATGAATG 480 CTAAGATAGC TGACTTCGGA CTCTCTAATA TGATGTCAGA TGGTGAATTT CTACGAACTA 540 GCTGTGGATC GCCAAATTAT GCAGCACCGG AGGTCATCTC AGGAAGGCTG TATGCGGGTC 600 CTGAGGTTGA TATCTGGAGC TGTGGTGTTA TCCTGTATGC CCTTCTCTGT GGCACCCTCC 660 CGTTCGACGA TGAGCACGTG CCTACGCTCT TTAAGAAGAT CCGAGGGGGT GTGTTCTACA 720 CGGAGTA TCTCAACCGT TCTATTGCCA CTCTGCTGAT GCACATGCTG CAGGTGGACC 780 CCTTGAAGCG AGCAACTATC AAAGACATAC GAGAGCATGA ATGGTTTAAA CAGGATTTGC 840 CCAGTTACCT CTTTCCTGAA GACCCCTCCT ATGATGCTAA CGTCATTGAT GATGAGGCTG 900 GAAAGAAGT ATGTGAAAAA TTTGAGTGTA CAGAATCAGA AGTGATGAAC AGTTTATACA 960 GTGGTGACCC TCAAGACCAG CTCGCAGTGG CTTATCATCT CATCATTGAC AATCGGAGAA 1020 TAATGAACCA AGCCAGTGAG TTCTACCTCG CCTCCAGTCC TCCAACGGGT TCCTTCATGG 1080 CGATATGGC CATGCACATT CCCCCCGGCC TGAAACCACA TCCTGAAAGG ATGCCACCTC 1140 TCATAGCAGA CAGCCCCAAA GCACGCTGTC CACTGGATGC ACTCAACACA ACTAAGCCCA 1200 AATCTTTAGC TGTGAAAAAA GCCAAGTGGC ACCTTGGGAT CCGAAGCCAG AGCAAACCAT 1260 ACGACATTAT GGCGGAGGTG TACCGAGCTA TGAAGCAGCT GGACTTTGAA TGGAAGGTAG 1320 TGAATGCATA CCATCTTCGA GTAAGAAGAA AAAACCCAGT GACTGGCAAT TACGTGAAAA 1380 TGAGCTTACA GCTTTACCTG GTTGACAATC GGAGCTATCT TCTAGACTTT AAAAGCATCG 1440 ATGATGAGGT GGTGGAGCAG AGGTCTGGTT CTTCAACACC TCAGCGCTCC TGTTCTGCTG 1550 CCGGCCTCCA CAGACCTCGG TCAAGTGTCG ATTCCAGCAC AGCCGAGAAC CATTCACTGT 1560 CTGGCTCTCT CACTGGTTCT TTGACTGGCA GAACTTTGTC CTCCGCTTCC CCGCGCCTGG 1620 GCAGTCATAC CATGGATTTT TTTGAAATGT GCGCCAGTCT TATCACTGCT TTAGCCCGTT 1680 GATAACCCAC CACCGGTCTC TGTCTTTCTG TTACCGCACT GTGAAATCAC ATACACTCTT 1740 CAAATTATTA CCGCACTCTC GGGTACCACA GGCTCTGCAA TAGAAGTTAT GTGAACATTC 1800 CCAGGTGACA TGCAGTGCTG CTGGAAACAC AGAAATCTGG CCTTCTGTTT ACTTTTAGAA 1860 :TCTGTAACT CTGCTGTGCC TATGATAGGT ATCAATAGCT AGGAACGGCT GAGTGCTGGT 1920 GAAGCTTGTT AACTTACACC CGTGAATTCA CTACACATGG TGAGCACACC TCACTGATGA 1980 ACCCGCTGAT CTCGGGGTGG TTCGGTGGGA CCGCCTTCCT TCACGTTTAG TTCATGTAAA 2040 ΓCCTGTTTGC CTCCTAAATT TCCATAGGTG TCAGGCTGTC TAGGCACTCT TGGACAAGAA 2100 3ATTCAGAAA TAGAGTAACT GTCAGTGAAA TATTATTTAA ATGTAGAAAT CCGAAATCCT 2160 :AA ATATCAGAAA CCAAAAGTCT TTTTTAATAC TTTCTGCAAA TACTGCCTAG TATTAGCCAT ACAGACTGTG TTTCTGATAA TAGGAGCCCA GTCTCCTAGC TTCCTGACAT 2280 TTGTGCAAGG CCCCTAGTTA AATCCCACTA CCACAAAGCA ATATAAAACC ATGCAAAGTT 2340 AGTGCATAGT TAAAGGAAAC AGGCAGATAC CAACAGTCTC TTAAAAGGAA ATCTATTCTT 2400 TGATCTCATT TGTGTTTATG AGACTGGGTA GCTGGGGGCT TGGGGGCGGG CTGGGAGTCA 2460 CAGCCCTTGG GCATCTTTGC TAATCCACTT GAACTTTGTT ATTGATGCCG AGTCCTCTCT 2520 CTTCCTCAGT AGCCACTGTC TTTGCTCATT GCTTTTCCCT TTTTTAACTC CTTTAGATCA 2580 AAACCTTGCT TTGGCCATAG GGTCTTTAAA TACTTTCAAA GCTTGATCTG CTGTGACCTT 2640 CACTGTTGAA CCTGATTGGA CAGGGAAGCC TTAAATATTT AAAAGTATAT TCTCCTGGAA 2700 TAACTACATG TGTTGTTTAC ATATATATAC ATATGTATTG ACACATCAGT GCTTTTAATC 2760 G 2761 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: P E N V (T/L) D A (Q/P) M N A X D A D F G (L) S N M 25 INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: E Y V (Y) G (K/L) (E) L F (A) Y L X K X G X X X X V 21 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: L Q V (D) P L K (R) A T I K (D) I X E (E) Ξ (W) F K Q 22 (2) INFORMATION FOR SΞQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: peptide (C) STRANDEDNESS : single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: P P L I A D X P K A R X P L D (A) L N (T) (T) K 21 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: peptide (C) STRANDEDNESS : single (D) TOPOLOGY: linear ixi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: K Q L D F Ξ (W/S) (K/G) K V V N A Y (H) L (R) V (R) X K X N 23 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: P E N V L T D A Q M N A X (D) A D F G L S N M 21 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCΞ DESCRIPTION: SEQ ID NO:31: (E) N V L (L/T) D A Q M N A X (D) A D F G L S N M 21 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: (E) F Y L A (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: (M) E Y V S G G E L F D Y I C K H G R V E E 21 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34 (M) P P L I A D S P K A R C P L D A L N T T K 22 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LΞNGTH: 21 amino acids (B) TYPE: peptide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: (M) K Q L D F E W K V V N A Y H L R V R R K 21 [ 2 ) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1783 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GATGGCTGAG AAGCAGAAGC ACGACGGGCG GGTGAAGATC GGACACTACG TGCTGGGCGA 60 CACGCTGGGC GTCGGCACCT TCGGCAAAGT GAAGATTGGA GAACATCAAT TAACAGGCCA 120 TAAAGTGGCA GTTAAAATCT TAAATAGACA GAAGATTCGC AGTTTAGATG TTGTTGGAAA 180 AATAAAACGA GAAATTCAAA ATCTAAAACT CTTTCGTCAT CCTCATATTA TCAAACTATA 240 CCAGGTGATC AGCACTCCAA CAGATTTTTT TATGGTAATG GAATATGTGT CTGGAGGTGA 300 ATTATTTGAC TACATCTGTA AGCATGGACG GGTTGAAGAG ATGGAAGCCA GGCGGCTCTT 360 TCAGCAGATT CTGTCTGCTG TGGATTACTG TCATAGGCAT ATGGTTGTTC ATCGAGACCT 420 GAAACCAGAG AATGTCCTGT TGGATGCACA CATGAATGCC AAGATAGCCG ATTTCGGATT 480 ATCTAATATG ATGTCAGATG GTGAATTTCT GAGAACTAGT TGCGGATCTC CAAATTATGC 540 AGCACCTGAA GTCATCTCAG GCAGATTGTA TGCAGGTCCT GAAGTTGATA TCTGGAGCTG 600 GGTGTTATC T GTATGCTC TCTTTGTGG CACCCTCCCA TTTGATGATG AGCATGTACC 660 TACGTTATTT AAGAAGATCC GAGGGGGTGT CTTTTATATC CCAGAATATC TCAATCGTTC 720 TGTCGCCACT CTCCTGATGC ATATGCTGCA GGTTGACCCA CTGAAACGAG CAACTATCAA 780 AGACATAAGA GAGCATGAAT GGTTTAAACA AGATTTGCCC AGTTACTTAT TTCCTGAAGA 840 CCCTTCCTAT GATGCTAACG TCATTGATGA TGAGGCTGTG AAAGAAGTGT GTGAAAAATT 900 TGAATGTACA GAATCAGAAG TAATGAACAG TTTATATAGT GGTGACCCTC AAGACCAGCT 960 TGCAGTGGCT TATCATCTTA TCATTGACAA TCGGAGAATA ATGAACCAAG CCAGTGAGTT 1020 CTATCTCGCC TCTAGTCCTC CATCTGGTTC TTTTATGGAT GATAGTGCCA TGCATATTCC 1080 CCCAGGCCTG AAACCTCATC CAGAAAGGAT GCCACCTCTT ATAGCAGACA GCCCCAAAGC 1140 AAGATGTCCA TTGGATGCAC TGAATACGAC TAAGCCCAAA TCTTTAGCTG TGAAAAAAGC 1200 CAAGTGGCAT CTTGGAATCC GAAGTCAGAG CAAACCGTAT GACATTATGG CTGAAGTTTA 1260 CCGAGCTATG AAGCAGCTGG ATTTTGAATG GAAGGTAGTG AATGCATACC ATCTTCGTGT 1320 AAGAAGAAAA AATCCAGTGA CTGGCAATTA CGTGAAAATG AGCTTACAAC TTTACCTGGT 1380 TGATAACAGG AGCTATCTTT TGGACTTTAA AAGCATTGAT GATGAAGTAG TGGAGCAGAG 1440 ATCTGGTTCC TCAACACCTC AGCGTTCCTG TTCTGCTGCT GGCTTACACA GACCAAGATC 1500 AAGTTTTGAT TCCACAACTG CAGAGAGCCA TTCACTTTCT GGCTCTCTCA CTGGCTCTTT 1560 GACCGGAAGC ACATTGTCTT CAGTTTCACC TCGCCTGGGC AGTCACACCA TGGATTTTTT 1620 TGAAATGTGT GCCAGTCTGA TTACTACTTT AGCCCGTTGA TCTGTCTCTA GTTTCTTTCT 1680 GTTATTGCAC TATGAAAATC AGTTATATTC TTTAAATTTT TATCTTACTT TTGGATAATA 1740 TCCATCCAGC TTGGACTTAA CCAGGCTGAA CTTGCTCAAA AGG 1783 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1736 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37 ATGGCTGAGA AGCAGAAGCA CGACGGGCGG GTGAAGATCG GACACTACGT GCTGGGCGAC 60 ACGCTGGGCG TCGGCACCTT CGGCAAAGTG AAGATTGGAG AACATCAATT AACAGGCCAT 120 AAAGTGGCAG TTAAAATCTT AAATAGACAG AAGATTCGCA GTTTAGATGT TGTTGGAAAA 180 ATAAAACGAG AAATTCAAAA TCTAAAACTC TTTCGTCATC CTCATATTAT CAAACTATAC 240 CAGGTGATCA GCACTCCAAC AGATTTTTTT ATGGTAATGG AATATGTGTC TGGAGGTGAA 300 TTATTTGACT ACATCTGTAA GCATGGACGG GTTGAAGAGA TGGAAGCCAG GCGGCTCTTT 360 CAGCAGATTC TGTCTGCTGT GGATTACTGT CATAGGCATA TGGTTGTTCA TCGAGACCTG 420 AAACCAGAGA ATGTCCTGTT GGATGCACAC ATGAATGCCA AGATAGCCGA TTTCGGATTA 480 TCTAATATGA TGTCAGATGG TGAATTTCTG AGAACTAGTT GCGGATCTCC AAATTATGCA 540 GCACCTGAAG TCATCTCAGG CAGATTGTAT GCAGGTCCTG AAGTTGATAT CTGGAGCTGT 600 GGTGTTATCT TGTATGCTCT TCTTTGTGGC ACCCTCCCAT TTGATGATGA GCATGTACCT 660 ACGTTATTTA AGAAGATCCG AGGGGGTGTC TTTTATATCC CAGAATATCT CAATCGTTCT 720 GTCGCCACTC TCCTGATGCA TATGCTGCAG GTTGACCCAC TGAAACGAGC AACTATCAAA 780 GACATAAGAG AGCATGAATG GTTTAAACAA GATTTGCCCA GTTACTTATT TCCTGAAGAC 840 CCTTCCTATG ATGCTAACGT CATTGATGAT GAGGCTGTGA AAGAAGTGTG TGAAAAATTT 900 CAATGTACAG AATCAGAAGT AATGAACAGT TTATATAGTG GTGACCCTCA AGACCAGCTT 960 GCAGTGGCTT ATCATCTTAT CATTGACAAT CGGAGAATAA TGAACCAAGC CAGTGAGTTC 1020 TATCTCGCCT CTAGTCCTCC ATCTGGTTCT TTTATGGATG ATAGTGCCAT GCATATTCCC 1080 CCAGGCCTGA AACCTCATCC AGAAAGGATG CCACCTCTTA TAGCAGACAG CCCCAAAGCA 1140 AGATGTCCAT TGGATGCACT GAATACGACT AAGCCCAAAT CTTTAGCTGT GAAAAAAGCC 1220 AAGTGGCATC TTGGAATCCG AAGTCAGAGC AAACCGTATG ACATTATGGC TGAAGTTTAC 1260 CGAGCTATGA AGCAGCTGGA TTTTGAATGG AAGGTAGTGA ATGCATACCA TCTTCGTGTA 1320 AGAAGAAAAA ATCCAGTGAC TGGCAATTAC GTGAAAATGA GCTTACAACT TTACCTGGTT 1380 GATAACAGGA GCTATCTTTT GGACTTTAAA AGCATTGATG ATGAAGTAGT GGAGCAGAGA 1440 :CTGGTTCCT CAACACCTCA GCGTTCCTGT TCTGCTGCTG GCTTACACAG ACCAAGATCA 1500 AGTTTTGATT CCACAACTGC AGAGAGCCAT TCACTTTCTG GCTCTCTCAC TGGCTCTTTG 1560 ACCGGAAGCA CATTGTCTTC AGTTTCACCT CGCCTGGGCA GTCACACCAT GGATTTTTTT 1620 GAAATGTGTG CCAGTCTGAT TACTACTTTA GCCCGTTGAT CTGTCTCTAG TTTCTTTCTG 1680 TTATTGCACT ATGAAAATCA GTTATATTCT TTAAATTTTT ATCTTACTTT TGGATA 1736 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1742 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38 ATGGCTGAGA AGCAGAAGCA CGACGGGCGT GTGAAGATCG GACACTACGT GCTGGGGGAC 60 ACCCTGGGCG TCGGCACCTT CGGCAAAGTG AAGATTGGAG AACATCAATT GACAGGCCAT 120 AAAGTGGCAG TTAAGATCTT AAATAGACAG AAGATTCGCA GTTTAGATGT TGTTGGAAAA 180 ATAAAACGAG AAATTCAAAA TCTTAAACTC TTTCGTCATC CTCATATTAT CAAACTCTAC 240 CAAGTGATCA GCACTCCAAC AGACTTTTTT ATGGTAATGG AATATGTGTC TGGAGGTGAA 300 TTGTTCGACT ACATCTGTAA ACACGGGAGG GTTGAAGAGG TGGAAGCTCG CCGGCTCTTC 360 CAGCAGATTC TGTCTGCCGT GGACTACTGT CACAGGCACA TGGTTGTCCA CAGGGACCTG 420 AAGCCAGAGA ACGTGTTGCT GGACGCCCAG ATGAATGCTA AGATAGCTGA CTTCGGACTC 480 TCTAATATGA TGTCAGATGG TGAATTTCTA CGAACTAGCT GTGGATCGCC AAATTATGCA 540 GCACCGGAGG TCATCTCAGG AAGGCTGTAT GCGGGTCCTG AGGTTGATAT CTGGAGCTGT 600 GGTGTTATCC TGTATGCCCT TCTCTGTGGC ACCCTCCCGT TCGACGATGA GCACGTGCCT 660 ACGCTCTTTA AGAAGATCCG AGGGGGTGTG TTCTACATCC CGGAGTATCT CAACCGTTCT 720 ATTGCCACTC TGCTGATGCA CATGCTGCAG GTGGACCCCT TGAAGCGAGC AACTATCAAA 780 GACATACGAG AGCATGAATG GTTTAAACAG GATTTGCCCA GTTACCTCTT TCCTGAAGAC 840 CCCTCCTATG ATGCTAACGT CATTGATGAT GAGGCTGTGA AAGAAGTATG TGAAAAATTT 900 GAGTGTACAG AATCAGAAGT GATGAACAGT TTATACAGTG GTGACCCTCA AGACCAGCTC 960 GCAGTGGCTT ATCATCTCAT CATTGACAAT CGGAGAATAA TGAACCAAGC CAGTGAGTTC 1020 TACCTCGCCT CCAGTCCTCC AACGGGTTCC TTCATGGACG ATATGGCCAT GCACATTCCC 1080 CCCGGCCTGA AACCACATCC TGAAAGGATG CCACCTCTCA TAGCAGACAG CCCCAAAGCA 1140 CGCTGTCCAC TGGATGCACT CAACACAACT AAGCCCAAAT CTTTAGCTGT GAAAAAAGCC 1200 AAGTGGCACC TTGGGATCCG AAGCCAGAGC AAACCATACG ACATTATGGC GGAGGTGTAC 1260 CGAGCTATGA AGCAGCTGGA CTTTGAATGG AAGGTAGTGA ATGCATACCA TCTTCGAGTA 1320 AGAAGAAAAA ACCCAGTGAC TGGCAATTAC GTGAAAATGA GCTTACAGCT TTACCTGGTT 1380 GACAATCGGA GCTATCTTCT AGACTTTAAA AGCATCGATG ATGAGGTGGT GGAGCAGAGG 1440 TCTGGTTCTT CAACACCTCA GCGCTCCTGT TCTGCTGCCG GCCTCCACAG ACCTCGGTCA 1500 AGTGTCGATT CCAGCACAGC CGAGAACCAT TCACTGTCTG GCTCTCTCAC TGGTTCTTTG 1560 ACTGGCAGAA CTTTGTCCTC CGCTTCCCCG CGCCTGGGCA GTCATACCAT GGATTTTTTT 1620 GAAATGTGCG CCAGTCTTAT CACTGCTTTA GCCCGTTGAT AACCCACCAC CGGTCTCTGT 1680 CTTTCTGTTA CCGCACTGTG AAATCACATA CACTCTTCAA ATTATTACCG CACTCTCGGG 1740 TA 1742 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 560 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38 M A E K Q K H D G R V K I G H Y V L G D T L G V G T F G K V K I G E H Q L T G H K V A V K I L N R Q K I R S L D V V G K I K R Ξ I Q N L K L F R H P H I I K L Y Q V I S T P T D F F M V M E Y S G G Ξ L F D Y I C H H G R V E E V Ξ A R R L F Q Q I L Ξ A V D Y , H R H M V V H R D L K P Ξ N V L L D A O M N A K I A D F G L S N M M S D G E F L R T S C G S P N Y A P Ξ V I S G R L Y A G P Ξ V D I S C G V I L Y A L L C G T L P F D D E H V P T L F K K I R G G V F Y I P E Y L N R S I A T L L M H M L Q V D P L K R A T I K D I R Ξ H E W F K Q D L P S Y L F P E D P S Y D A N V I D D E A V K E V C E K F E C T E S E V M N S L Y S G D P Q D Q L A V A Y H L I I D N R R I M N Q A S E F Y L A S S P P T G S F M D D M A M H I P P G L K P H P E M P P L I A D S P K A R C P L D A L N T T K P K S L A V K K A K W H L G I R S Q S K P Y D I M A E V Y R A M K O L D F E W K V V N A Y H L R V R R K N P V T G N Y V K M S L Q L Y L V D N R S Y L L D F K S I D D E V V E Q R S G S S T P Q R S C S A A G L H R P R S S V D S S T A E N H S L S G S L T G S L T G S T L S S A S P R L G S H T M D F F E M C A S L I T A L A R 560

Claims: Claims : 1. A cDNA encoding a mammalian AMP protein kinase. 2. A cDNA encoding a human AMP protein kinase. 3. A cDNA as claimed in claim 2 and as set out in Table 5. 4. A cDNA encoding a rat AMP protein kinase. 5. A cDNA as claimed in claim 4 and as set out in Table 1. 6. A polynucleotide of at least 6 nucleotides which hybridises selectively to a cDNA encoding a mammalian AMP protein kinase as claimed in any one of claims 1-5 (including complementary sequences) . 7. A polynucleotide probe which comprises all, or any convenient fragment, of a cDNA as claimed in any one of claims 1-5. 8. A synthetic polypeptide of at least 6 amino acids and corresponding to a cDNA as claimed in any one of claims 1-5 9. A recombinant mammalian AMP protein kinase. 10. A recombinant human AMP protein kinase. 11. A recombinant rat AMP protein kinase. 12. Antibodies raised to a synthetic polypeptide as claimed in claim 7. 13. Antibodies raised to a recombinant mammalian AMP protein kinase as claimed in any one of claims 8-10.

Inventors: BERI RAJINDER KUMAR, CARLING DAVID, FORDER ROBERT ANTHONY, BERI, RAJINDER, KUMAR, CARLING, DAVID, FORDER, ROBERT, ANTHONY
Applicants: BERI RAJINDER KUMAR, CARLING DAVID, FORDER ROBERT ANTHONY, ZENECA LTD, BERI, RAJINDER, KUMAR, CARLING, DAVID, FORDER, ROBERT, ANTHONY, ZENECA LIMITED
Application year: 1994
Main classification: C12N9
IPC codes: C07K7, C07K14, C07K16, C12N5, C12N9, C12N15, C12P21, C12Q1, C12R1